many different solutions to a problem

derivative project that removes proprietary software

little-known facts better known as

Indeed, I'd heard of hosts.allowed but would have never known that they were part of a package/system called tcpwrappers (which I don't think I've ever heard of).

what does this mean exactly?

How easy is this to do? Is this an example that shows a negative? Is it good for an issue like this to show the negatives and positives? Are there more positives than negatives---if so, what does this show?

Author Response:

Reviewer #3:

Maintaining the balance between stem cell proliferation and cell differentiation is an essential challenge of all stem cell niches. In the shoot apical meristem of plants, these functions are spatially separated into the central zone and peripheral zone, respectively. How these zones communicate to give rise to proper stem cell behavior has been a research focus for many years.

In this manuscript, the authors suggest that the small secreted peptide CLE40 and the receptor kinase like protein BAM1 form a novel pathway that contributes to meristem homeostasis by stimulating the expression of the central stem cell inducer WUSCHEL primarily from the meristem periphery. Importantly, this pathway acts antagonistically to the well-studied CLV pathway, which is only active in the center of the meristem and is molecularly highly similar to the CLE40/BAM1 system. This model is experimentally supported mainly by analysis of spatial localization patterns in the meristem using transcriptional and translational reporters and by the analysis of genetic interactions.

The findings of the authors are novel, highly relevant and would certainly be of great interest for the plant community. However, the manuscript could be substantially improved to provide better support for the conclusions laid out.

Of major concern are the reporter genes and imaging data: Partial colocalization and exclusion from CZ and OC are one of the main arguments of the authors to claim that CLE40/BAM1 function together and antagonistically to CLV3/CLV1 in controlling WUS expression.

Working with reporters as proxies for endogenous gene expression needs to be backed up by proper controls. Given the central importance of the reporters for the conclusions it is essential to show that the regulatory sequences used for the CLE40 reporter are sufficient to rescue a cle40 mutant.

We show now in a new supplemental figure (1) the expression patterns of two different CLE40 reporter lines (differening in length of the promoter region) in the root, which are identical, and (2) that expression CLE40 from the CLE40 promoter rescues the cle40 mutant root phenotypes, which were described in earlier work. See Fig2-SupplFig. 1

It is essential to show that ... the observed expression of the reporter is consistent across the majority of different T1 lines and, most importantly, that the pattern reported here is consistent with in situ data for endogenous CLE40 mRNA.

RNA in situ analysis is difficult due to the low expression level of CLE40, and the small size of the CLE40 transcript. We show in Fig2-SupplFig2 expression data for 4 independent transgenic CLE40 reporter lines, confirming the general conclusions that we present in this manuscript.

The authors have previously published in situs for CLE40 that do not show the exclusion from the CZ and OC (Hobe et al., 2003, Figure 2a,c), which urgently needs clarification.

The RNA expression data from Hobe et al. are displayed at low mag and low resolution, and might have suffered from high background.

Figures 2, 4 and 5 show imaged meristems in great detail but each focus only on a single sample. I strongly recommend to also include quantitative data on multiple samples to substantiate the claims. This could be likely be done with standard software, such as MorphographX.

The data we showed before represented typical examples from a wide range of data that we analysed. All original data are being made publicly available for reanalysis. We have now added multiple examples from multiple samples, and also added quantitative data from fluorescence analysis. See new Supplementary Fig2-SupplFig. 2, Fig.4-SupplFig. 1, Fig.4-SupplFig. 2, Fig.4-SupplFig. 3, Fig.5-SupplFig. 1, Fig.5-SupplFig. 2, Fig.5-SupplFig. 3, Fig.5-SupplFig. 4

Whereas the inhibitory effect of WUS on CLE40 is convincingly shown using ectopic WUS expression and the hypomorphic wus7 allele (Figure 2) the quantification of WUS positive cells in Figure 7 is problematic. Although it was done over multiple samples it heavily relies on manual scoring, which is prone to bias. The same is true for the width/height measurements of different meristems. An unbiased computational image analysis would certainly give more reliable results.

We are grateful for this suggestion. We normally analyse samples in an anonymised manner. We have now also quantified the number of WUS positive cells using the Imaris software, as suggested, see Fig.7-SupplFig.1, and found that this analysis supported our previous conclusions. We also added a figure showing multiple samples from this experiment. See new Supplementary Fig7-SupplFig. 2

One major point that the authors try to establish is that the CLE40 signal that eventually leads to reduction in meristem size is transduced via the BAM1 receptor. However, only genetic interactions, which are complicated by intricate feedbacks, are show to substantiate this claim. For a strong statement on CLE40/BAM1 ligand/receptor interactions, advanced imaging technologies available to the authors or biochemical experiments would be necessary.

We are currently not aware of a reliable and applicable experimental approach that would allow us to show direct interaction of the CLE40 peptide with its receptors in vivo. Biochemical experiments using purified peptides and/or receptors are, so far, contradictory: Shinohara et al. (2015) used chemically synthesized arabinosylated CLV3 peptide and photoaffinity labelling to show binding of CLV3 to a BAM1-Halo-TAG fusion protein expressed in BY-2 cells. However, using BAM1 protein purified from insect cell lines which was biotinylated in the Creoptix WAVE system, Crook et al. (2020) found no significant binding activity for synthetic CLV3 peptide. Our preliminary conclusion from these data sets is that binding of peptides to receptors should be best evaluated in vivo, since important posttransciptional and posttranslational modifications, as well as coreceptors, can strongly modify peptide-receptor interactions.

We have here added data showing that in the root, BAM1 receptor but not CLV1 is required for CLE40 dependent regulation of root meristem development, indicating again that CLE40 and BAM1 are likely to act in the same signaling pathway throughout development. See new Supplementary Fig6-SupplFig. 1

Similarly, the genetic studies need some clarification: The authors show that cle40 and bam1 single mutants as well as cle40/bam1 double mutants all show a comparable reduction in meristem size, suggesting epistasis. In contrast, a reduction in meristem size can not be observed if cle40 is combined with clv1, which according to the proposed model appears to be unexpected. The interpretation of the genetic experiments is complicated by the well-known fact that BAM1 expression is regulated by the CLV pathway and loss of CLV signaling leads to ectopic expression of BAM1 in the OC which can partially compensate for the loss of CLV1, due to the molecular similarity of the two receptors. The shift of BAM1 expression from the PZ towards the OC could explain why there is no significant reduction in meristem size since CLE40 induced signaling at the PZ would be inhibited by the lack of the BAM1 receptor. To clarify the specific interaction of CLE40 with BAM1 and/or CLV1 the authors could try to restore BAM1 levels in the PZ of cle40/clv1 mutants by expressing BAM1-GFP from an appropriate promoter (e.g. RPS5 or UBQ10). This experiment would allow to distinguish between the genetic interaction of CLE40 with CLV1 from the feedback between CLV1 and BAM1 expression.

The suggested experiment, to misexpress BAM1 from the RPS5 or UBQ10 promoter, is not feasible, since this results generally in a much higher expression level, which, in our hands, is not "tolerated" by CLV-family receptors. We found that higher level expression of RLKs generally causes mislocalisation of nonfunctional proteins.

Overall, the manuscript could be strengthened by inclusion of additional molecular data probing the directness of WUS inhibiting CLE40 and/or BAM1 expression.

We are planning to set-up experiments for detailed studies on the transciptional regulation of genes in the stem cell control pathways, and will in the future also investigate the feedback regulation of WUS onto CLE40 and BAM1. However, such analysis goes far beyond the scope of our current manuscript.

Hypothesis: Businesses can use emotional branding to keep up with the times.

NOTE: Colourette Cosmetics is a Filipino makeup brand that also promotes inclusivity. It's cosmetics is geared towards the younger generation no matter the gender.

the world is through the will, connects back to the idea that mother believes the world is run by will not love

similar to harrison believing that she can will herself to nto care about the world

SciScore for 10.1101/2021.09.14.21263603: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: All study participants provided written informed consent and all study protocols were approved by the Cedars-Sinai Medical Center institutional</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coupling efficiency was confirmed by incubation of 625 beads from each coupled region with a phycoerythrin-conjugated anti-6×□HisTag antibody (Abcam, Cambridge, UK) at a concentration of 10 μg/mL for 45 min shaking at 900 rpm and room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-6×□HisTag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The bound AABs were detected by addition of R-phycoerythrin-labelled goat anti-human IgG detection antibody (Ab) (5□µg/ml, Dianova, Hamburg, Germany) for 1 hr at RT, after several PBS washes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Other antigens were produced in-house using E.coli SCS1 carrying plasmid pSE111, which contains an N-terminally located hexa-histidine-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSE111</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For all HCW participants, EDTA plasma specimens were transported within 1 hour of phlebotomy to the Cedars-Sinai Department of Pathology and Laboratory Medicine and underwent serology testing using the Abbott Diagnostics SARS-CoV-2 IgG chemiluminescent microparticle immunoassay (Abbott Diagnostics, Abbott Park, Illinois) against the nucleocapsid (N) antigen of the SARS-CoV-2 virus (12, 13).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott</div><div>suggested: (Abbott, RRID:SCR_010477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses: Data processing and analysis were performed using R v3.5.1 and KNIME 2.12 (https://www.knime.org/) (21, 22).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>KNIME</div><div>suggested: (Knime, RRID:SCR_006164)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.09.17.460782: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Detection was achieved using horseradish peroxidase-conjugate secondary antibody anti-rabbit and anti-mouse (Bio Rad #1706516, #1706515) and visualized with ECL (Cytiva RPN2232).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (Bio-Rad Cat# 170-6516, RRID:AB_11125547)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies: The primary antibodies used in this study are: rabbit anti-SARS-CoV-2 Spike S1 Subunit (Sino Biological, 40150-T62)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibody used are: horseradish peroxidase-conjugate anti-rabbit and anti-mouse (Bio Rad #1706516, #1706515).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Bio-Rad Cat# 170-6515, RRID:AB_11125142)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, AP-conjugated goat anti-rat IgG antibody (SIGMA A8438) or AP-conjugated goat anti-rabbit IgG antibody (SIGMA A8025) was added, and the plates were further incubated for 1 hour at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rat IgG</div><div>suggested: (Sigma-Aldrich Cat# A8438, RRID:AB_258391)</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: (Sigma-Aldrich Cat# A8025, RRID:AB_258372)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FACS: Vero E6 cells were incubated with RBD protein (0.45 μg/mL, final concentration) followed by incubation with human anti-RBD antibody (primary antibody) (40150-D003, Sino Biological) and goat anti-human IgG AF488-conjugated antibody (secondary antibody) (A-11013, Thermo Fisher Scientific)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Molecular Probes Cat# A-11013, RRID:AB_141360)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hi-5 cells (BTI-TN-5B1-4) (Gibco #B85502) were cultured in Express Five™ SFM (Serum-Free Media) medium (Gibco #B85502 Expression Systems) at a cell density of 0.5 x 106 cells/mL and infected with recombinant virus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Hi-5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FACS: Vero E6 cells were incubated with RBD protein (0.45 μg/mL, final concentration) followed by incubation with human anti-RBD antibody (primary antibody) (40150-D003, Sino Biological) and goat anti-human IgG AF488-conjugated antibody (secondary antibody) (A-11013, Thermo Fisher Scientific)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a wash step in 1× kinetic buffer for 120 s, the ACE2-Fc-captured biosensor tips were then submerged for 300 s in wells containing different concentrations of antigen (RBD E. coli, insect, and HEK-293) to evaluate association curves, followed by 900 s of dissociation time in kinetic buffer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD protein production in E. coli: The SARS-CoV-2 Spike Receptor Binding Domain sequence (aa 319-541, Uniprot ID P0DTC2) was cloned with a C-terminal 6x-His tag into a pET-21a(+) plasmid. E. coli BL21 Star™ (DE3) (genotype: F−ompT hsdSB (rB−, mB−) galdcmrne131) competent cells, and E. coli Lemo21 (DE3) (genotype: fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS/ pLemo(CamR)) competent cells were transformed with 100 ng of plasmid of interest.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-21a(+</div><div>suggested: RRID:Addgene_12669)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD protein production in insect cells: The SARS-CoV-2 Spike Receptor Binding Domain sequence (aa 319-541, Uniprot ID P0DTC2) was cloned into a pFAST-bac1 plasmid downstream of the gp64 signal sequence to promote secretion, along with a C-terminal 8x-His tag for affinity purification.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFAST-bac1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Raw data were analyzed using the Biopharma Finder 2.1 software from ThermoFisher Scientific.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Biopharma Finder</div><div>suggested: None</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

However, the high costs (resources), the time-consuming production, the requirement of specific equipment, and access to dedicated facilities could be a limitation for many laboratories or for the industrial production. By contrast, the bacterial-derived RBD offers a low production cost, a broader availability, and easy handling as main advantages, which make it more accessible. However, limitations in the quality of the produced sample include the absence of glycosylation that partially affects protein stability and efficiency, the presence of heterogeneous folded populations, and the relative low production yields which may result in a final product not eligible for some clinical and medical applications. Overall, all the recombinantly produced RBDs represent valuable tools for research purposes against the pandemic. Recently, expression and purification strategies described in this article have been also proved to be successful in the production of mutants of RBD corresponding to the variants of concern.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.09.15.460454: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: All cell lines have tested negative for mycoplasma contamination.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines used in this study: 293FT cells were obtained from Dr. Kosuke Yusa’s Lab. 293FT.Cas9 cell lines were generated through lentiviral integration of an EF1a-Cas9-T2A-BlastR construct at low MOI to achieve single-copy integration.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT</div><div>suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate cell lines permissive to Spike-Pseudotyped lentiviral infection, 293FT.Cas9 cells were engineered to stably express SARS-CoV-2 receptors ACE2 and TMPRSS2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT.Cas9</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For in vitro transcribed mRNA, 293T cells were transfected for functional testing using Lipofectamine messengerMAX (Invitrogen) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293FT.Cas9.ACE2/TMPRSS2 clonal cell lines were harvested by trypsinization and resuspend at a density of 70.000 cells per 30 μL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT.Cas9.ACE2/TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro transcription of S protein mRNA: Templates for in vitro transcription were generated by cloning P1 and P13 between NcoI and NotI sites of pTNT-B18R-6His (addgene plasmid 58979, a kind gift from Steven Dowdy (30)).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTNT-B18R-6His</div><div>suggested: RRID:Addgene_58979)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For transfection, 1 μg of lentiviral transfer vector (pCSGW-GFP), were mixed with 0.72 μg of gag-pol expressing plasmid p8.9 and 68.33 fmol of S protein expressing construct in 500 μL of optiMEM media followed by the addition of 2 μL of PLUS reagent and incubation for 5 minutes at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCSGW-GFP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>p8.9</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These PCR products were both visualized on an agarose gel as well as TA-cloned using ‘TA Cloning Kit with pCR2.1 vector and OneShot TOP10 Chemically Competent E.coli’ (ThermoFisher) according to kit instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCR2.1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The template used to align the ALKBH5 reads to was constructed manually, by performing in silico Gateway cloning, inserting the ALKBH5 CDS and mRuby3 CDS into the pLIX_403 vector (Addgene plasmid #41395).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLIX_403</div><div>suggested: RRID:Addgene_41395)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Full DNA sequences of these plasmids are found at a Zenodo provided doi: 10.5281/zenodo.5470001. “Wuhan” in plasmid names refers to the S protein DNA sequence from the Wuhan-Hu-1 isolate (Genbank: MN908947.3) while “18F” refers to the removal of the last 18 amino acids of the S protein C terminus (ER retention sequence) and the addition of a FLAG tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zenodo</div><div>suggested: (ZENODO, RRID:SCR_004129)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data was analysed using FlowJo software (BD Biosciences) and displayed as % cells infected at 1:500 dilution of pseudotyped virus, normalized to the intronless construct infection rates (Figure 7).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Staining was developed using 20X LumiGLO® Reagent and 20X Peroxide reagents according to manufacturer’s recommendations (Cell Signaling Technology, #7003). cDNA analysis: RNA was extracted from the frozen cell pellets using RNeasy Mini Kit (Qiagen) and treated with ezDNase (ThermoFisher) before applying oligo(dT) guided 1st strand cDNA synthesis using SuperScript IV reverse transcriptase (ThermoFisher), all according to manufactures’ recommendations.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher</div><div>suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All reads were mapped back to the original construct DNA sequence using SnapGene software to assess individual mRNA splicing events.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After another round of bead purification, samples were pooled and submitted to Edinburgh Genomics, where they were further processed, barcoded and run on PromethION platform.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PromethION</div><div>suggested: (PromethION, RRID:SCR_017987)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

The main limitation lies in the quantification of the observed events in comparison to the reads without events which can be influenced by size dependency of some of the sample preparation steps as the full-size S protein cDNA is over 4kb long and many of the observed splicing events can be shorter than 200 bp and depleted by many sample preparation methods. Reported here are distributions based on the full length read population alone within each sample, but care should be applied before comparing these frequencies to other datasets with different sample preparation methods, such as the ChAdOx1 Nanopore RNA direct data (Sup Figure 8). Hundreds of randomly spaced splicing events characterized by the cDNA direct sequencing approach for the S protein and similar events seen in other proteins highlight the fact that even when the transgene’s CDS is apparently well designed and expression of full-length protein can be detected, the status quo design is just not optimal for RNA expression as transcript heterogeneity will inevitably impact both product levels (yield) and homogeneity. The impact of this will be dependent on the nature of the product, whether it is expressed in vitro or in vivo. In vitro expression offers opportunities to improve homogeneity by application of purification methods. Two thirds of cryptically spliced RNA molecules would be out-of-frame and would not just impact protein yield but any translation in vivo would generate novel peptides with potential immuno...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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Results from scite Reference Check: We found no unreliable references.

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

"what's it called?"

his tag can be labeled in lysate

small tag may not alter the conformation and function of the protein

2 points:

1. binding Kd is about 1nM
2. Kd for different targets could be slightly different

SciScore for 10.1101/2021.09.14.460356: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">g, anti-Human antibody was added to the wells and incubated for 1 hour followed by six washes as before.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Human</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD-SD1, wild type RBD and mutant RBD domains were subcloned into pcDNA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RBD-SD1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One day before transfection, CHO-S cells were seeded at a density of 1 x 106 cells/mL in 45 mL culture flask.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO-S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lipofectamine® transient transfection of RBD constructs: For transient expression of RBD-SD1, RBD wild-type and RBD mutants, 293T cells were cultured and and incubated at at 37℃ with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 neutralization assay: All aspects of the assay utilizing virus were performed in a BSL3 containment facility according to the ISMMS Conventional Biocontainment Facility SOPs for SARS-CoV-2 cell culture studies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation with 293TACE2 cells for 48 hours at 37°C, cells were washed twice with PBS, lysed with Luciferase Cell Culture Lysis 5x reagent (Promega), and NanoLuc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293TACE2</div><div>suggested: RRID:CVCL_YZ65)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 Spike ECD 1-1208 (682-GSAS-685; 986-PP-987) fused to the T4 fibritin trimerization domain with C-terminal Avi- and His-tag were synthesized with gene block (IDT) and cloned into pcDNA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA</div><div>suggested: RRID:Addgene_66792)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">On the day of transfection, 75 µL of FectoPRO® transfection reagent (PolyPlus-transfection®) was mixed with 5 mL of 15 µg/mL pcDNA3 plasmid DNA harboring antibody encoding sequence in CD-CHO media and incubated for 10 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3</div><div>suggested: RRID:Addgene_15475)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The atomic models and cryo-EM maps generated for the N-612-017, N-612-014, and N-612-004 Fabs complexed with SARS-CoV-2 S have been deposited at the PDB (http://www.rcsb.org/) and the Electron Microscopy Databank (EMDB) (http://www.emdataresource.org/) under accession codes 7S0C, 7S0D, 7S0E and EMD-24786, EMD-24787, EMD-24788, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>http://www.emdataresource.org/</div><div>suggested: (EMDataResource.org, RRID:SCR_003207)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Half-maximal inhibitory concentrations (IC50 values) for mAbs were determined using 4-parameter nonlinear regression (Prism, GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Movies were collected using a 3x3 beam image shift pattern with SerialEM automated data collection software (Mastronarde, 2005) at a nominal magnification of 45,000x (super-resolution 0.4345 Å/pixel) using a defocus range of −0.7 to −2.0 µm.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For all datasets, movies were patch motion corrected for beam-induced motion including dose-weighting within cryoSPARC v3.1 (Punjani et al.,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coordinates were rigid body and B-factor refined in PHENIX v1.19 (Adams et al., 2010) followed by sequence matching and repeated cycles of phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">refine and manual building in Coot (v0.9.3) (Emsley et al., 2010) (Table S6)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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Reply to the reviewers

Full Revision

Manuscript number: RC-2021-00785

Corresponding author: Christian, G. Specht

1. General Statements

Dear Editor,

We greatly appreciate the reviewers’ constructive comments on our manuscript ‘Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses’. We were particularly pleased that all four reviewers agreed that our data yield new insights into the structure of inhibitory glycinergic synapses, and represent both a technical and conceptual advance the field of synaptic neuroscience.

The reviewers have consistently raised one main criticism, namely the use of endogenously expressed GlyRs tagged with the fluorescent protein mEos4b, which could potentially have an impact on receptor expression, trafficking and function. We have addressed this point by performing whole-cell recordings of GlyR currents in cultured neurons that show that glycinergic transmission and therefore function is preserved. We have also addressed all other comments of the reviewers in the revised manuscript, including a thorough revision of the text and the addition of new data and figures as detailed in the point-by-point response.

Point-by-point description of the revisions

Reviewer 1:

Summary:

In this manuscript Maynard et al describe a newly generated knockin mouse to study the endogenous distribution of Gly receptors in the spinal cord. Using quantitative confocal imaging and SMLM the distribution and levels of GlyRs at spinal cord synapses is compared between dorsal and ventral horn. They found that levels of synaptic GlyR are higher in dorsal than ventral spinal cord synapses. Nevertheless, the ratio to gephyrin seems constant, except for synapses in superficial layers of the dorsal horn, where gephyrin levels exceeded the levels of GlyRs. There are also fewer, but larger synapses in the ventral horn than in the dorsal horn. These findings are further corroborated by an SR-CLEM approach. Furthermore, it is shown that in a mouse model for hyperekplexia GlyR levels are lower, but still enriched at synapses, and the dorsal-ventral gradient in GlyR expression was maintained. The difference in size of ventral and dorsal synapses observed in WT animals was also lost in the oscillator mouse, suggesting that particularly the ventral synapses are affected. Despite these differences, the density of GlyRs per synapse remained similar.

Major comments:

Line 113: "labeling the_ _b__-subunit has proven difficult". This statement is unclear and it would be informative for readers to grasp what exactly has been difficult, and why the approach described here overcomes that? Related to that, the authors state "KI animals reach adulthood and display no overt phenotype, suggesting that the presence of the N-terminal fluorophore does not affect receptor expression and function". That is indeed reassuring, but it does not exclude that receptor numbers, function and distribution are altered. As it seems there is no prior literature on tagging the beta subunit, additional evidence that the tag does not interfere with receptor trafficking or functioning would be desirable

We have clarified why it has been difficult to label the GlyR beta subunit until now, lines 113-115 _“To date, labeling of GlyRβ in situ using immunocytochemistry has proven difficult due to a lack of reliable antibodies that recognize the native β-subunit (only antibodies for Western blotting recognizing the denatured protein are available), which has severely limited the study of the receptor.”_ Hence it was important to us to generate this knock-in mouse in order to study the endogenous GlyR at synapses, which is the least well studied receptor mediating fast synaptic transmission.

The reviewer makes an important point regarding the labeling of the GlyRβ-subunit with a fluorescent protein that has also been raised by the other reviewers. We have now verified receptor function by patch clamp recordings of glycine currents in whole-cell configuration in spinal cord neuron cultures from the mEos4b KI mouse (new Supplementary Fig. S2C). At saturating glycine concentrations of 300 μM we found no difference in chloride influx between mEos4 KI and WT mice. Since glycine concentrations in the synaptic cleft are in the millimolar range during synaptic transmission, these data strongly suggest that glycinergic transmission is not affected by the presence of the mEos4b under physiological conditions, despite a minor shift in the EC50.

There are several other strong arguments that suggest that mEos4b-GlyRb expression, subcellular localization and function are the same as those of the native subunit. Firstly, the mEos4b sequence was inserted after the signal peptide and before the beginning of the coding sequence of the mature β-subunit (Fig. S1). Since the mEos4b sequence does not interrupt the coding sequence it is less likely to affect the receptor conformation. Secondly, we did not notice any behavioural phenotypes in animals carrying the GlrbEos allele. At the time of weaning, the genotypes of the pups corresponded to the expected Mendelian frequency (new Fig. S2A). Moreover, we did not observe a reduction in live expectancy of GlrbEos/Eos animals (new Fig. S2B), demonstrating that the mEos4b-GlyRb does not cause pathology in older animals.

Most importantly, our imaging data (Fig. 1-3) provide exhaustive evidence that mEos4b-GlyRb assembles with GlyR alpha subunits as heteropentameric receptor complexes that are trafficked to the plasma membrane and inserted into the synaptic membrane due to their interaction with the gephyrin scaffold at functional synapses. Using quantitative imaging, we have also shown that homozygous GlrbEos/Eos KI mice have exactly twice the number of receptors at synapses as heterozygous animals, strongly suggesting no interference in receptor trafficking to the plasma membrane and gephyrin binding. As the mEos4b mice were also bred with the oscillator mouse model of hyperekplexia, which is lethal when homozygous, we could further test the combined effect of GlrbEos and GlyRa1spt-ot. The presence of both alleles did not lead to any noticeable phenotypes in heterozygous oscillator mice. On the contrary, both synaptic targeting and the packing density of the receptors were not altered in this model, despite a region-specific reduction in synapse size due to the reduced availability of the intact GlyRa1 subunit.

We believe that these data overwhelmingly support our conclusion that the presence of the mEos4b tag does not alter the structure and function of the receptor, making this mouse model uniquely suited to study the dynamics and regulation of glycinergic synapses in a quantitative manner and at the molecular level.

In the Discussion the authors conclude that "Our quantitative SR-CLEM data lend support to the first model, whereby inhibitory PSDs in the spinal cord are composed of sub-domains that shape the distribution of the GlyRs". This conclusion seems however based on one example image in Fig 3G that is not very convincing. The EM image seems to show two clearly separated PSDs opposed by two distinct active zones. So, although this conclusion is of high interest, more support should be given to substantiate this conclusion. More general, these subsynaptic domains (SSDs) are hardly further explored, but seem relevant for transmission, particularly given that the synaptic pool of GlyRs at these synapses is not saturated by single release events. How general are these SSDs at these synapses?

The representative image in Fig. 3G shows two SSDs within the same postsynaptic site with a continuous presynaptic active zone. It should be noted that the PALM/SRRF images were taken of the entire 2 µm thick slice, whereas the electron micrograph shows only a single 70 nm section. We verified throughout the full 3D stack of serial sections that the presynaptic site remains continuous, which it does. We would also like to point out the scale of the image showing that the two SSDs are only around 170 nm apart, i.e. spatially very close. Our conclusions are however not based on this single image but the whole dataset. The graph in Fig. 3I shows 3 synapses (out of N = 36), in which the GlyR density at separate SSDs could be quantified, demonstrating that the receptor density is not different between SSDs. The reviewer is correct that we do not further analyse the SSDs beyond their density and the analysis of the segmentation of the postsynaptic sites (Fig. 3E-G). Further work on the functional role of SSDs in synaptic transmission is outside the scope of this manuscript and would indeed merit future study.

The approach for counting molecules based on the PALM acquisition has been developed in prior publications and seems robust. It would however be worth to present the reader with a bit more background and explain the assumptions of this approach in more detail. Particularly, since counting of mEos4b can be problematic, as there are multiple dark and fluorescent states of this fluorophore that could be influenced by the illumination scheme, see for instance De Zitter et al., Nat Methods 2019. Since the preceding SRRF acquisition already exposes the fluorophore to high and continuous 561-nm laser power this could skew the counting due to unaccounted conversion and perhaps bleaching of mEos4b. In line with this, although throughout the manuscript the term 'absolute copy numbers' is used the reported numbers are at best an estimate based on a number of assumptions. I think the wording 'absolute numbers' is therefore deceiving and should be nuanced.

We have clarified how the molecule conversion is calculated (Fig. S7 legend), to provide a more complete description of the way in which the values were obtained. Further we have explained how we calculated the probability of detection. Since the probability of detection accounts for any unconverted or non-functional mEos4b molecules, our molecule counting approach is relatively resistant to potential pre-bleaching of fluorophores. It should be noted, that 561 nm illumination had no obvious effect on the non-converted (green) mEos4b fluorophores, as judged by the fact that the intensity of receptor puncta was unaffected by the SRRF recordings. We appreciate the reviewers point regarding the term ‘absolute copy number’ and we have adjusted our wording throughout the manuscript accordingly.

Related, most of the quantifications are in estimating the number of receptors, and not so much the distribution with the PSD. The term "molecular arrangement" - also used in the title - might therefore be misleading, there is in fact little characterization of how GlyRs are placed within the PSD. More focused analysis quantifying the distribution of receptors within the PSD and/or SSDs would strengthen the manuscript.

By estimating the number of receptors and the exact size of synapses, the main conclusion of our study is that receptor density at dorsal and ventral synapses is identical, independent of synapse size, subdomains, or in fact loss of GlyRs in a mouse model of hyperekplexia. This observation clearly relates to how receptors are packed within synapses, and thus describes their molecular arrangement.

The reported N is confusing and makes it hard to judge the reproducibility of the data. Sometimes it refers to number of images, sometimes number of synapses, but it is unclear from how many experiments these are drawn. This should be reported more completely (number of animals should be reported at least) and consistently. In figure 1, the N numbers (N=3-5 images) are particularly low and question how consistent these findings are across multiple animals.

We have clarified the N in the figure legends, to reflect the full size of the datasets that have been analysed.

The levels of mRFP-Gephyrin seem to differ between the different mouse lines, is this a significant difference?

No significant differences in mRFP-gephyrin levels were found in animals with different mEos4b-GlyRb genotype (Fig. 1B). However, expression of mRFP-gephyrin in heterozygous animals is 50% of that in homozygous mRFP-gephyrin KI animals (not shown).

The ICQ analysis for co-localization is hardly explained. How do we interpret this parameter? What does an average value of ~0.3 mean? A comparison with sets of proteins that do not overlap as a negative control would strengthen the conclusion.

We have clarified that an ICQ value of 0.3 is indicative of a very high spatial correlation between pixels, and provided a corresponding reference for ICQ analysis (lines 209-210). We would like to point out that the scale of the ICQ is between -0.5 to 0.5, meaning that a value of 0.3 comes close to complete correlation.

Minor comments:

Very little fluorescence was detected in the forebrain, despite the high reported expression of the Glrb transcript". Can the authors expand on this? What would explain this discrepancy?

We have clarified the text to include “suggesting that protein levels are controlled by post-translational mechanisms in a region-specific manner, as previously proposed (Weltzien et al., 2012)” (Lines 152-153). The reason for this discrepancy is not known. However, the distribution of mEos4b expression throughout the brain is as expected, based on the literature.

"What region is quantified in Fig 1B? is the same region in all conditions? This should be specified more clearly as the manuscripts presents a clear gradient in expression levels in the spinal cord and thus the location will influence the intensity measurements.

We have explained in the text that this is the region at the centre of the ventral horn identified by the white square in Fig. 1A, and that the same region was analysed for all images across all animals. Page 5, lines 160-161 “The same region of the ventral horn, indicated by the white square in Fig. 1A was taken for quantification of mEos4b-GlyRβ and mRFP-gephyrin expression in all conditions.”

The labeling approach does not differentiate between surface and internal receptors, this should be made more explicit in the text.

Whilst this is correct, we have only analysed mEos4b-positive synapses that had corresponding gephyrin clusters, meaning synapses where receptors are located in the postsynaptic membrane. Indeed we found that all mEos4b clusters imaged colocalised with mRFP-gephyrin clusters. We have adjusted the text accordingly, page 6, line 205-206 “All mEos4b-GlyR clusters closely matched the mRFP-gephyrin clusters, confirming the localization of the receptors in the postsynaptic membrane.”

Significance:

The presented data are interesting and the experiments are technically advanced and carefully performed. Particularly the SR-CLEM approach is technically advanced. The datasets present a quantitatively detailed characterization of spinal cord synapses and will be of interest for researchers working in the field of spinal cord circuitry, as well as super-resolution imaging. The conceptual advance for the field is however somewhat limited. It seems that the presented data confirm the general notion that receptor numbers and synapse size are highly correlated. So, although this manuscript describes very interesting observations, in its present form the manuscript does not provide any new mechanistic insight or significant advance in our understanding of how these synapses operate.

We thank the reviewer for his/her comments relating to the technicality of our manuscript. However we think that the statement “The conceptual advance for the field is however somewhat limited” is unfair, as this level of organisation of inhibitory synapses at the molecular scale has never been achieved before, as pointed out by the other reviewers, and especially not as regards different ages of animals and a disease model that directly affects receptor numbers in a region-specific manner. We therefore believe that our study will have a substantial impact within the fields of synaptic neuroscience as well as quantitative neurobiology.

Referee cross-commenting:

I agree with the other reviewers that this study is technically advanced, but I remain critical towards the extent of conceptual advancement this study brings and there are some important concerns with the presented data that need to be addressed. Nevertheless, indeed many of these concerns can be addressed without additional experiments. As pointed out also by other reviewers additional validation that the fusion proteins are not disrupting their function or organization would be important.

Reviewer 2:

Summary:

Maynard et al. investigate (inhibitory) glycinergic synapses in mouse spinal cord, which regulate motor and sensory processes. The authors analyse the molecular architecture and ultra-structure of these synapses in native spinal cord tissue using quantitative super-resolution correlative light and electron microscopy. The major finding is that GlyRs exhibit equal receptor-scaffold occupancy and constant absolute packing densities across the spinal cord and throughout adulthood, although ventral and dorsal inhibitory synapses differ in size. Moreover, what the authors call a „stereotypic arrangement" is even maintained in a hypomorphic mutant (oscillator), which is deficient in the adult GlyR a1 subunit.

Specific comments:

To reach their conclusions the authors generate two knock-in mouse lines, one with mEOS-labelled GlyR ß-subunit and one with mRFP-labelled gephyrin, a subsynaptic scaffolding protein of inhibitory synapses, which are subsequently crossed. Both changes are not unproblematic, as mutations in the N-terminal end of the GlyR ß subunit polypeptide chain might interfere with the assembly of functional GlyR (consisting of a und ß subunits) and and mutations at the N-terminal end of gephyrin interfere with it's homo-oligomerization into higher molecular assemblies.

We have demonstrated that the function of mEos4b-GlyRb does not differ significantly from WT GlyRs, by carrying out electrophysiological experiments (new Fig. S2C). For a detailed response, please see the response to the first comment of reviewer 1. The mRFP-gephyrin KI strain has been validated and published previously (see Machado et al., 2011, J Neurosci; Specht et al. 2013 , Neuron) and was not specifically generated for this study. The experiments with the oscillator mutant did not include the mRFP-gephyrin allele. In these experiments, the wildtype GlrbEos/Eos (Fig. 4, 5) behaves exactly as the GlrbEos/Eos in the double knock-in (Fig. 1, 2), further validating the mouse models used.

However, in this experimental design both labelled proteins reach postsynaptic membrane specialisations. In case of the ß-subunit quantitative evaluation confirms that heterozygous animals contain only half of the labelled protein as homozygous, which is an indication but not a proof that the correct stoichometry of adult GlyR is maintained. Likewise, mRFP-labelled gephyrin assembles with WT-gephyrin in subsynaptic domains, but it is not clear, if the size and density of the synapses is changed by the knock-in procedure as compared to WT-synapses.

An effect of the mRFP tag on gephyrin clustering can be ruled out, since we observed no difference in synapse size and receptor density in GlrbEos/Eos animals with (Fig. 1, 2) and without the GphnmRFP allele (Fig. 4, 5, oscillator wild-type controls). Similarly, the synaptic mEos4b-GlyRb levels in heterozygous animals were precisely half those of the homozygous animals, strongly suggesting that the expression and trafficking of the tagged receptor subunit is unchanged, as the reviewer acknowledges. In the absence of any obvious behavioural and/or functional phenotypes (Fig. S2) this KI model is in our view is an exceptional tool to study GlyRs expressed at endogenous levels in a cell-type specific manner.

Accepting these constraints, which to the knowledge of this reviewer have never been addressed to satisfaction, the authors provide a technically excellent, comprehensive analysis of glycinergic synapses in the spinal cord of double knock-in mice. Therefore, it should be stated in the title, that the investigations were performed with double knock-in instead of „native" spinal cord. Text and figures are clear and accurate and represent the state of the art.

We thank the reviewer for the positive comments regarding the techniques used in the study, and the clarity of the text and figures. We have adjusted the title as requested.

Finally, the reviewer would like to raise a minor point: the term postsynaptic density is derived from electron microscopical studies of synapses, where asymmetrical synapses display a „postsynaptic density" but symmetrical synapses do not. The latter were identified as inhibitory synapses and therefore, by definition, inhibitory synapses do not have a postsynaptic density, but rather a postsynaptic membrane specialisation. The use of the term „postsynaptic density" should, therefore, be restricted to excitatory synapses.

We are conscious of the importance of correct definitions and have revised the terminology, referring to “postsynaptic sites”, “postsynaptic domains”, and “postsynaptic specializations” as appropriate throughout the manuscript.

Significance:

The authors provide a state of the art advanced light and electron microscopical analysis of glycinergic synapses in the mouse spinal cord. They suggest a robust "stereotypical" mechanism in place, which guarantees a fixed stoichiometry of relevant components, which is even maintained in a hypomorphic mutant, which is believed to represent a mouse model of human hyperekplexia (startle disease).

Referee cross-commenting:

I would like to corroborate the arguments of the previous reviewer: it is not clear to which extent the fusion proteins influence the measurements, which are technically very advanced and well done, however. The authors do definitely not investigate "native spinal cord" as stated in the title.

The argument concerning fusion proteins must be taken especially serious as the fusions were induced in regions known to be responsible for assembly of glycine receptors and oligomerization of gephyrin.

We have verified the receptor function with electrophysiological recordings and clarified exactly where the fluorescent protein was inserted (see reviewer 1 response). Given the similarity in synapse size, fluorescence intensities and molecule densities observed in neurons expressing different combinations of tagged and native receptors and scaffold proteins, we strongly believe that all animal models used are well suited to the experimental aims of our study.

Reviewer 3:

Summary:

Glycinergic synapses are the least well understood of synapses that mediate fast synaptic transmission. The manuscript by Maynard et al. adds new information about the structural aspects of these synapses, using PALM and EM imaging of spinal cord synapses from mice at 2 and 10 months. The authors created a knock-in mouse that expresses a tagged GlyRbeta subunit, allowing synaptic localization of glycine receptors; all synaptically localized glycine receptors are thought to require the beta subunit to be tethered by gephyrin. The authors compare synaptic profiles from: 2 month old vs. 10 month old mice; dorsal vs. ventral horn; and GlyR1-reduced vs. wild type mice. Strikingly, they find a tight relationship across all of these variables between glycine receptor puncta and gephyrin puncta, as well as an apparently constant "packing density" of glycine receptors. They conclude that synaptic extent is likely to be the most important determinant of synaptic strength, as the density of receptors within the postsynaptic density is constant. These results use cutting-edge imaging and are analyzed with care, and add new information to our understanding of these relatively less well characterized synapses._

Major comments:

The key conclusions are convincing and the claims appear solid. Additional experiments are not needed to support these claims. The data and the methods are largely presented in such a way that they can be reproduced, although there are minor suggestions for improvement below.

We thank the reviewer for his/her positive comments.

Minor comments:

Do the authors have any comment on the requirement during, e.g. LTP, for insertion of a gephyrin-GlyR unit? The lead author has speculated that gephyrin creates "slots" for GlyRs; yet apparently each slot is already filled in the snapshots taken here. How might postsynaptic LTP occur (Kandler group, Kauer group papers)?

Given the reciprocity of GlyR and gephyrin clustering at synapses, the occupancy of binding sites (and in turn the number of available ‘slots’) is dependent on the strength of receptor-scaffold interactions, as discussed previously (Specht 2020, Neuropharmacol). In this study we demonstrate that the density of GlyRs at synapses is constant, which implies that the receptor occupancy is also the same, with the possible exception of mixed inhibitory synapses in the superficial dorsal horn that contain a majority of GABAARs. The PALM/SRRF data are represented as rendered image reconstructions and not as pointillist representations, and the detection of unoccupied binding sites is below the spatial resolution of our approach. However, the high spatial correlation of the signal intensities (ICQ ≈ 0.3) suggests that receptor occupancy is equal between and within synapses. It has previously been established that there are more scaffold proteins than receptors at synapses (Specht et al. 2013, Neuron; Patrizio et al. 2017, Sci Rep). Based on these studies we report that approximately half the gephyrin binding sites are occupied by receptors (lines 262-655). We have also expanded the discussion, describing how shape and size of synapses may affect synaptic transmission, as well as the possible role of receptor-gephyrin interactions in synaptic plasticity at glycinergic synapses.

It would be very interesting in the discussion to contrast the present observations with what is known about excitatory synapses (NMDA and AMPAR distributions) and GABAergic synapses. Are the authors at all surprised that receptor packing is constant across conditions? Can the authors speculate on how non-gephyrin binding receptors (homomeric alpha receptors, which are found in recordings) may function and be tethered to the membrane.

We have included additional information about receptor numbers and distributions at excitatory (lines 428-438) and GABAergic (lines 389-393) synapses in the discussion. So far, homomeric GlyRs composed of alpha subunits have been found to be exclusively extrasynaptic. As stated on page 4, lines 111-112 the beta subunit is required for binding of the GlyR to gephyrin and subsequent anchoring at the synapse. Previous studies have shown exocytosis of receptors to occur at extrasynaptic sites followed by lateral diffusion to synapses. Homomeric GlyRs are therefore most likely targeted to the extrasynaptic plasma membrane where they remain due to the lack of the beta subunit.

Figure S1. It would be most helpful to quantify this; at the least to include an atlas-like drawing to allow identification of the structures illustrated and containing Glrb; better yet would be quantification of staining in regions where this is strongest.

We have added an atlas indicating the different brain regions expressing mEos4b-GlyRb protein as a new Supplementary Fig. S3. The regional expression pattern agrees with the available literature about protein expression of the GlyRb subunit in different brain regions and hence provides further evidence that mEos4b-GlyRb is expressed like the native receptor. Due to the relatively low resolution of the tiled image no accurate quantification was possible. We have however added higher magnification confocal images of representative brain regions expressing varying amounts of GlyRb.

The fact that the lower panel in B is labeled as +/+ across all groups is initially confusing; perhaps relabel as mEos4 -/-, +/- and +/+?

We assume that the reviewer is referring to Fig1B. The genotype of both the GlrbEos and the GphnmRFP allele is now indicated on the x-axes, and the legend has been modified to clarify that all these animals were homozygous for GphnmRFP/mRFP. We have strived to remain consistent throughout the manuscript when referring to genotypes and protein levels.

Do gephyrin levels drop in WT mice as well as in the mEosr-GlyRb mouse between 2 and 10 months? Do the authors have any thoughts on this (Supp figure S2)?

We found no differences in gephyrin levels between 2 and 10 months. Fig. S2 (now Fig. S4C) shows the number of synaptic gephyrin clusters, which was the same at different ages and genotypes.

Significance:

Glycinergic synapses are the least well understood of synapses that mediate fast synaptic transmission. The manuscript by Maynard et al. adds new information about the structural aspects of these synapses, using PALM and EM imaging of spinal cord synapses from mice at 2 and 10 months. The authors created a knock-in mouse that expresses a tagged GlyRbeta subunit, allowing synaptic localization of glycine receptors.

This will be of interest to those studying inhibitory synapses, and more broadly to synaptic morphologists, physiologists and imagers for comparison with other synapse types.

My own expertise is NOT in these techniques, but I am a synaptic physiologist with a standing interest in glycinergic synapses; thus I am not providing serious technical critiques.

Referee cross-commenting:

Hi all, I agree with the other two reviewers, and do not have anything else to add.

Reviewer 4:

Summary:

The authors used a correlative approach and combined photo-activated localization microscopy with electron microscopy to characterise Glycinergic synapses in spinal cord tissue. Some of the major findings are:

• The receptor-scaffold occupancy and packing densities of glycinergic synapses in different regions of the spinal cord are the same.
• Gephyrin clusters in the spinal cord are composed of sub-domains that shape the GlyR clusters.
• Ventral horn synapses are generally larger, more complex (containing a number of gaps) and contain more GlyRs. -In a mouse model of Hyperekplexia, the number of GlyRs is reduced resulting in smaller synapses in the ventral spinal cord.

Major comments:

Are the key conclusions convincing? Yes

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. No

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. N/A

Are the data and the methods presented in such a way that they can be reproduced? Yes

Are the experiments adequately replicated and statistical analysis adequate? Yes

Minor comments:

Specific experimental issues that are easily addressable. Please see below

Are prior studies referenced appropriately? Yes

Are the text and figures clear and accurate? Yes

Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Please see below.

As the authors pointed out, fusing mEos to the extrasynaptic terminal of GlyRb has been difficult and therefore this construct would benefit the larger scientific community. Fig 1C is a nice imaging control for expression efficiency, however, it is in stark contrast with the lack of functional control. Do authors have any electrophysiological evidence showing that the insertion of mEos4b doesn't modulate channel function? I would assume that the construct would be tested in cell lines before the KI mouse line was created. Was any functional analysis done? If yes, it would be very useful to show it. I do appreciate that the authors used a standard insertion between the 4th and 5th AA in the extracellular domain, which in most cases does not abolish channel function. Given the lack of an obvious phenotype in the KI mouse model, I believe that this is also the case here. However, I disagree with the statement in lines 120-121: "the presence of the N-terminal fluorophore does not affect receptor expression and function." I believe that if there are no electrophysiological measurements of GlyR function, this statement remains speculative. As the authors pointed out in their previous publication: "receptor function and gephyrin binding are not independent properties. Instead, we think that conformational changes triggered at extracellular or intracellular protein domains have downstream consequences on channel opening as well as receptor clustering." In line with this, my concern is that the modulation of channel function by mEos4b could result in an altered cluster size at synapses. There is a large body of literature showing that just one missense mutation in the extracellular domain of ion channel subunits can lead to synaptopathies because the channel function gets modulated, and there is an abundance of similar examples involving mutations of GlyR and GABAAR subunits. In my view, comparing the function of GlyRs incorporating wt-GlyRb and mEos4b-GlyRb subunits is important for the correct interpretation of the main findings of this work and would strengthen the publications.

As the reviewer points out, the insertion of the mEos4b sequence was considered carefully in order to have the least impact on receptor function. GlyR channelopathies are often caused by point mutations within the coding sequence, which is not the case in the GlrbEos allele. Instead, the mEos4b sequence was inserted after the single peptide of GlyRb, duplicating several amino acid residues in order to maintain the correct cleavage site and N-terminus of the mature receptor, and to not interrupt the GlyRb coding sequence (Fig. S1B). In order to verify that the mEos4b-tag does not affect GlyR function, we have now carried out electrophysiological experiments (new Fig. 2C). For a detailed description please see the response to the first comment of reviewer 1.

Line 189: Are the authors making conclusions based on intensity comparison of red mEos4b and mRFP? The title of this section implies that the red form of mEos was compared to mRFP(?) But mEos converts from green to red only partially. Was the probability for conversion taken into account at this point? Please clarify which version of mEos was compared to mRFP._

In line 189 (now 218) we compared the intensities of mRFP-gephyrin with those of converted (red) mEos4b in SRRF / PALM super-resolution images of the synapses (Fig. 2D). Since the absolute intensities are altered by the process of image reconstruction, the probability that mEos4b is photoconverted does not have to be taken into account. The constant ratio of the SRRF and PALM image intensities confirms the data in Fig. 1D showing that GlyR and gephyrin amounts are highly correlated throughout the spinal cord (with the exception of the superficial layers of the dorsal horn). We have clarified in the text that this analysis was carried out on reconstructed SRRF images of mRFP-gephyrin and PALM images of mEos4, line 202.

Line 192: Please clarify how the density threshold was calculated/determined? This is important for the replication of the experiments, and it also has implications for the calculated probability of detection of mEos4b. I am not aware that this probability was calculated before for mEos4b and therefore other researchers may decide to rely on the value calculated here.

We have now clarified in more detail how the probability of detection was calculated (new Supplementary Fig. S7 legend).

In Fig. 2 Gephyrin clusters look consistently smaller than GlyR clusters, which is inconsistent with the published work. I assume that the difference in size is a consequence of different image reconstruction methods(?) However, I would assume that SRRF would have lower resolution than your PALM measurements and that would result in wider Gephyrin clusters. Could you please explain this discrepancy? Also, could you provide an estimate for the image resolution in SRRF and PALM techniques? For SMLM, localization precision would suffice.

We have provided an estimate of the resolution of the two techniques using Fourier ring correlation, which gave 46 nm for SRRF and 21 nm for PALM. Additionally we have precised the discrepancy between reconstruction methods, page 6, lines 194-200 “The spatial resolution was estimated using Fourier ring correlation (FRC), which measures the similarity of two images as a function of spatial frequency by comparing the odd and even frames of the raw image sequence. According to this analysis, the spatial resolution of SRRF was 46 nm and that of PALM 21 nm. It should be noted that the synaptic puncta in the SRRF images appear somewhat smaller and brighter due to differences in the reconstruction methods that result in differences in the dynamic intensity range.”

Why is the data in Fig. 5D and E represented as Detections/Synapse instead of GlyRs/Synapse? Could you please re-plot this so that a comparison with Fig. 2H and I is straightforward?

We have converted the detections to receptor copy numbers as requested (Fig. 5D,E).

Figure S5C: for P=0.5, 2=0.25. Please correct. Also, I assume that the second graph is what would be observed experimentally for dimers and P=0.5. Please clarify in the figure caption.

This was a mistake and has been corrected. We have also clarified which parts of the calculations are theoretical and which values were derived from our experimental data. We have provided a more detailed description in the figure legend of Supplementary Fig. S7.

Line 606: Please provide a complete derivation of this formula.

We have provided a full derivation of this formula (new Fig. S7C).

Significance:

The work described here seem to be a natural progression of a publication by Patrizio et al., 2017 that came out from the same laboratory. This study uses advanced methodologies in the imaging space to visualise and characterise Glycinergic synapses in spinal cord tissue. The experiments described here are technically demanding as evidenced by the relatively small number of publications describing super-resolution measurements in tissue samples. Even more rare are studies that attempt to do single protein counting in neuronal culture and tissue sections. Therefore, I believe that this work brings significant technical advancement in the field of super-resolution and corelative microscopy. The findings are also highly significant for all fields of neuroscience in which the structure of inhibitory Glycinergic synapse is relevant, ranging from the fundamental understanding of inhibitory synapse function to pathologies involving Glycinergic signalling._

I have substantial experience in different microscopy methods, including quantitative super-resolution microscopy based on single molecule counting. My background also covers the structure and function of GABAA and Glycine receptors using electrophysiology. I am familiar with the methods used in electron microscopy and the process of creating KI mouse lines, however I don't have hands-on experience in these fields._

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this manuscript Maynard et al describe a newly generated knockin mouse to study the endogenous distribution of Gly receptors in the spinal cord. Using quantitative confocal imaging and SMLM the distribution and levels of GlyRs at spinal cord synapses is compared between dorsal and ventral horn. They found that levels of synaptic GlyR are higher in dorsal than ventral spinal cord synapses. Nevertheless, the ratio to gephyrin seems constant, except for synapses in superficial layers of the dorsal horn, where gephyrin levels exceeded the levels of GlyRs. There are also fewer, but larger synapses in the ventral horn than in the dorsal horn. These findings are further corroborated by an SR-CLEM approach. Furthermore, it is shown that in a mouse model for hyperekplexia GlyR levels are lower, but still enriched at synapses, and the dorsal-ventral gradient in GlyR expression was maintained. The difference in size of ventral and dorsal synapses observed in WT animals was also lost in the oscillator mouse, suggesting that particularly the ventral synapses are affected. Despite these differences, the density of GlyRs per synapse remained similar.

Major comments:

• Line 113: "labeling the -subunit has proven difficult". This statement is unclear and it would be informative for readers to grasp what exactly has been difficult, and why the approach described here overcomes that? Related to that, the authors state "KI animals reach adulthood and display no overt phenotype, suggesting that the presence of the N-terminal fluorophore does not affect receptor expression and function". That is indeed reassuring, but it does not exclude that receptor numbers, function and distribution are altered. As it seems there is no prior literature on tagging the beta subunit, additional evidence that the tag does not interfere with receptor trafficking or functioning would be desirable
• In the Discussion the authors conclude that "Our quantitative SR-CLEM data lend support to the first model, whereby inhibitory PSDs in the spinal cord are composed of sub-domains that shape the distribution of the GlyRs". This conclusion seems however based on one example image in Fig 3G that is not very convincing. The EM image seems to show two clearly separated PSDs opposed by two distinct active zones. So, although this conclusion is of high interest, more support should be given to substantiate this conclusion. More general, these subsynaptic domains (SSDs) are hardly further explored, but seem relevant for transmission, particularly given that the synaptic pool of GlyRs at these synapses is not saturated by single release events. How general are these SSDs at these synapses?
• The approach for counting molecules based on the PALM acquisition has been developed in prior publications and seems robust. It would however be worth to present the reader with a bit more background and explain the assumptions of this approach in more detail. Particularly, since counting of mEos4b can be problematic, as there are multiple dark and fluorescent states of this fluorophore that could be influenced by the illumination scheme, see for instance De Zitter et al., Nat Methods 2019. Since the preceding SRRF acquisition already exposes the fluorophore to high and continuous 561-nm laser power this could skew the counting due to unaccounted conversion and perhaps bleaching of mEos4b. In line with this, although throughout the manuscript the term 'absolute copy numbers' is used the reported numbers are at best an estimate based on a number of assumptions. I think the wording 'absolute numbers' is therefore deceiving and should be nuanced.
• Related, most of the quantifications are in estimating the number of receptors, and not so much the distribution with the PSD. The term "molecular arrangement" - also used in the title - might therefore be misleading, there is in fact little characterization of how GlyRs are placed within the PSD. More focused analysis quantifying the distribution of receptors within the PSD and/or SSDs would strengthen the manuscript.
• The reported N is confusing and makes it hard to judge the reproducibility of the data. Sometimes it refers to number of images, sometimes number of synapses, but it is unclear from how many experiments these are drawn. This should be reported more completely (number of animals should be reported at least) and consistently. In figure 1, the N numbers (N=3-5 images) are particularly low and question how consistent these findings are across multiple animals.
• The levels of mRFP-Gephyrin seem to differ between the different mouse lines, is this a significant difference?
• The ICQ analysis for co-localization is hardly explained. How do we interpret this parameter? What does an average value of ~0.3 mean? A comparison with sets of proteins that do not overlap as a negative control would strengthen the conclusion.

Minor comments:

• "Very little fluorescence was detected in the forebrain, despite the high reported expression of the Glrb transcript". Can the authors expand on this? What would explain this discrepancy?
• What region is quantified in Fig 1B? is the same region in all conditions? This should be specified more clearly as the manuscripts presents a clear gradient in expression levels in the spinal cord and thus the location will influence the intensity measurements.
• The labeling approach does not differentiate between surface and internal receptors, this should be made more explicit in the text.

Significance

The presented data are interesting and the experiments are technically advanced and carefully performed. Particularly the SR-CLEM approach is technically advanced. The datasets present a quantitatively detailed characterization of spinal cord synapses and will be of interest for researchers working in the field of spinal cord circuitry, as well as super-resolution imaging. The conceptual advance for the field is however somewhat limited. It seems that the presented data confirm the general notion that receptor numbers and synapse size are highly correlated. So, although this manuscript describes very interesting observations, in its present form the manuscript does not provide any new mechanistic insight or significant advance in our understanding of how these synapses operate.

Referee Cross-commenting

I agree with the other reviewers that this study is technically advanced, but I remain critical towards the extent of conceptual advancement this study brings and there are some important concerns with the presented data that need to be addressed. Nevertheless, indeed many of these concerns can be addressed without additional experiments. As pointed out also by other reviewers additional validation that the fusion proteins are not disrupting their function or organization would be important.

SciScore for 10.1101/2021.09.03.21263105: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: All participants provided written informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Study participants and sample collection: The study participants were enrolled in the ACTIV-2/AIDS Clinical Trials Group (ACTG) A5401 phase 2 randomized, placebo-controlled trial of bamlanivimab 7000mg and 700mg mAb therapy.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgG was detected by incubation with MSD SULFO-TAG anti-IgG antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 quantitative Laboratory Developed Test (LDT) was developed utilizing open mode functionality on m2000sp/rt (Abbott, Chicago, IL) by using EUA Abbott SARS-CoV-2 qualitative reagents30.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott</div><div>suggested: (Abbott, RRID:SCR_010477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Raw sequence data were analyzed using PASeq v1.4 (https://www.paseq.org).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PASeq</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, data were quality filtered using Trimmomatic (v0.30), using a Q25/5 bp sliding window and a 70 bp minimum length.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Trimmomatic</div><div>suggested: (Trimmomatic, RRID:SCR_011848)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Filtered reads were then merged with pear v0.9.6 aligned to the reference sequence using Bowtie2 v2.1.0)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pear</div><div>suggested: (PEAR, RRID:SCR_003776)</div></div><div style="margin-bottom:8px"><div>Bowtie2</div><div>suggested: (Bowtie 2, RRID:SCR_016368)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were performed in GraphPad Prism (Version 9.1.1)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

One limitation to this study is that bamlanivimab is no longer used clinically as a single agent. These results, though, provide important proof of principle for the role that drug resistance may have on virologic and clinical efficacy of SARS-CoV-2 antiviral therapies. These lessons have implications for the development of novel COVID-19 antiviral therapies and have continued relevance for other clinically-approved single agent monoclonal antibody treatments29 and for combination therapies where one agent may be ineffective due to circulating variants13. Another limitation of this study is the limited sample size of this phase 2 study, especially in the bamlanivimab 7000mg cohort. While treatment-emergent mutations were not found in ACTIV-2 participants receiving the higher 7000mg dose of bamlanivimab, they were frequently detected in the larger BLAZE-1 phase 2 trial of the 7000mg dose7. One difference between these studies was the longer duration since symptom onset for the ACTIV-2 participants, who enrolled a median of 6 days since symptom onset versus 4 days for the BLAZE-1 participants. This likely led to higher pretreatment viral loads, which we found to be a risk factor for resistance emergence. Unfortunately, baseline viral loads could not be compared between studies as the BLAZE-1 study did not use a quantitative SARS-CoV-2 viral load assay. These disparate results highlight the importance of incorporating quantitative viral load testing and resistance testing for CO...

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04518410</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">ACTIV-2: A Study for Outpatients With COVID-19</td></tr></table>

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.09.07.21262911: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study was approved by the ethics and research committees of the Instituto Nacional de Medicina Genómica (CEI/1479/20 and CEI 2020/21).<br>Field Sample Permit: Effective reproduction number estimation for variants B.1.1.222 and B.1.1.519: We grouped all sequenced samples based on the epidemiological week as the date of sample collection.<br>Consent: Verbal consent and identification were the first steps when calling each subject included in the final analysis.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequencing was performed on the MinION platform, and the final library (15 ng) was loaded onto the flow cell R.9 according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MinION</div><div>suggested: (MinION, RRID:SCR_017985)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A final consensus FASTA file was generated by first marking positions not covered by at least 20 reads from either group as low coverage and building a pre-consensus FASTA with BCFtools consensus, which was subsequently aligned against the reference sequence using muscle (v.3.8.1551).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BCFtools</div><div>suggested: (SAMtools/BCFtools, RRID:SCR_005227)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phylogenetic Analysis: The sequences were aligned with MAFFT (version 7.475) using the FFT-NS-2 algorithm20,21.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A maximum-likelihood phylogeny was calculated with FastTree (version 2.1.11) compiled with the double precision tag using a generalized time-reversible model (GTR)22,23.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FastTree</div><div>suggested: (FastTree, RRID:SCR_015501)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

Hypo.thesis apparently can't handle page 96, so here are my notes on it. New note: "'Or complex,' volleyed Dunraven. 'Remember the universe.' Funny banter isn't something I've seen in many other Borges stories, but he's good at it. New note: "volleyed Dunraven." Borges breaks the rules a bit by using a descriptive dialogue tag. I think he does it really well, though. "Volleyed" communicates more than, say, "retorted." It tells us a bit about Dunraven's tone and what kind of comment he's making, which is already clear because Borges' dialogue is sharp and clear, but it also tells us that Dunraven might think of conversation as a game or a competition. Originality helps here, too. I don't think I've seen "volleyed" used as a dialogue tag anywhere else. New note: "Climbing up steep sandy hills...." Their walk needs a little more specificity, in my opinion. Maybe it would be more clear with a description of these characters coming over a hill past Dunraven's family shack and seeing the labyrinth in the distance, or something like that. The beginning of this paragraph was pretty disorienting at first. The disorientation definitely could be intentional, though. Borges could be setting something up with that vagueness.

SciScore for 10.1101/2021.09.12.459978: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids of all S variants were transiently expressed in HEK293 Freestyle cells with polyethylenimine.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, transient expression of recombinant ACE2 was achieved in Expi293 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus neutralization assay: The pseudovirus neutralization assays were performed using 293T cells overexpressing ACE2 as described previously32.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of S-Beta, Gamma, Delta, and Kappa variants: The codon-optimized DNA sequences corresponding to residues 1-1209 of S-Beta, Gamma, Delta, and Kappa variants were individually cloned into the mammalian expression vector pcDNA3.4-TOPO (Invitrogen, U. S. A.), which contains a foldon trimerization domain based on phage T4 fibritin followed by a c-Myc epitope and a hexa-repeat histidine tag as previously described32,42.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4-TOPO</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data processing was accomplished by using cryoSPARC v2.14.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data acquisition was performed on a 300 keV Titan Krios microscope equipped with a Gatan K3 direct electron detector (Gatan, U. S. A.) in a super-resolution mode using EPU v2.10 software (ThermoFisher Scientific, U. S. A.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>EPU</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction: All 2x binned super-resolution movie files were analyzed by Relion-3.044 with dose-weighting and 5×5 patch-based alignment using MotionCor2 (v1.2.6)45.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MotionCor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural visualization and rendering of structural representations were accomplished by using UCSF-ChimeraX and Pymol 2.4.1 (Schrodinger Inc. U. S. A.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Pymol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50 was determined by a four-parameter logistic regression using GraphPad Prism 9 (GraphPad, U. S. A.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

.readwise-whatis

.tag-why .read-howto

.c1 .tag-whyto Use both Keyword and Categorical tags.

.c2

.tag-howto

.c2

SciScore for 10.1101/2021.09.08.21263027: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: This study was approved by the local Ethical Review Board (41/21) and conducted according to the Declaration of Helsinki.<br>Consent: Blood Plasma samples of patients with PIMS/MIS-C according were taken after written informed consent in the department of Pediatric Cardiology of the Saarland University Hospital (Homburg/Saar, Germany), the Department of Pediatrics, Klinikum Saarbrücken (Germany), the Department of Pediatric Rheumatology and Immunology of the University Children’s Hospital Muenster (Germany), the Department of Pediatrics, Hospital Sant Joan de</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All patients with PIMS/MIS-C fulfilled the WHO criteria, in addition all patients were seropositive for antibodies against SARS-CoV-2 or had positive PCR, with the exception of one patient who had only reported contact to SARS-CoV2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SARS-CoV2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ELISA for autoantibodies against PGRN, IL-1-Ra: The ELISA for autoantibodies was performed as previously described (19).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PGRN</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant IL-1-Ra at 40ng/mL (Biozol, #PPT-AF-2000-01RA) alone or with either anti-IL-1-Ra antibody at 5 µg/mL (antibodies-online#ABIN2856394), recombinant SLP-antibody at 5µg/mL (abcam, #ab191883), plasma diluted 1:20 of a patient with acute PIMS/MIS-C and high-titered IL-1-Ra-antibodies (PIMS-I), or plasma diluted 1:20 of the same patient 7 months after PIMS/MIS-C without anymore detectable IL-1-Ra-Abs were preincubated for 2h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#PPT-AF-2000-01RA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IL-1-Ra</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>antibodies-online#ABIN2856394</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In short, the antigens were obtained using the coding sequences of the GRN gene encoding PGRN, isoform 1 precursor of IL1RN were recombinantly expressed with a C-terminal FLAG-tag in HEK293 cells under the control of a cytomegalovirus promoter (pSFI).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

The relatively small number of cases included in the present study, due to the rarity of the disease, is a limitation. Nonetheless, these antibodies suggest to be pathogenetically relevant and should be further investigated in PIMS/MIS-C and other hyperinflammatory diseases.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

manual action time to think

.h1 Can I write the Summary Note using the tag 'dot h1'?

Probably a false distinction, because "packages" is used in a way that it implies a distinction from "gems", when in actuality

1. gems are packages, too (Ruby packages)
2. it's referring specifically to JavaScript/node/npm packages,

... so there is only truly a distinctio if you are specific enough to say JavaScript packages.

Author response

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September 9, 2021

We would like to thank ASAPbio for selecting our preprint for review! We are excited to contribute to this new process and hope others will find it as helpful as we have. The comments generated by the “crowd” were detailed and thoughtful. Below we respond to the major discussion points and if there were specific reviewer comments relevant to the discussion point, we also included that statement. We also responded to each specific comment. We would love to continue this discussion, so we invite further feedback and responses! Thanks so much for your time.

-Chelsea Kidwell, Joey Casalini, and Minna Roh-Johnson

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Major Discussion Point #1:One of the most important claims is that mitochondria are the organelles responsible for the activation of the signals of cell proliferation. However, a previous report by the last author reported that macrophages transfer cytoplasm to recipient cells. It cannot be excluded that other organelles or cellular fragments are transferred as well and contribute to the observed effects (ERK activity). Perhaps a good way to solve this would be the use of macrophages that are devoid of mitochondria. At least, this aspect should be discussed in the manuscript.

🡪 We had first considered two approaches to test the requirement and sufficiency of macrophage mitochondria in cancer cell proliferation. The first was to generate rho-zero macrophages (mtDNA-deficient), as you mention in your comment, such that the macrophages did not have functional mitochondria. However, we use primary human macrophages for all of our studies, and these cells would not survive long enough to generate rho-zero cells (which requires that the cells be treated with low levels of ethidium bromide for weeks). The second is to biochemically purify mitochondria from macrophages and directly inject these mitochondrial preps into breast cancer cells. We actually did this experiment, and cancer cells injected with purified mitochondrial preps exhibited higher proliferation (by live timelapse microscopy) compared to control cells. However, we also found that the mitochondrial purifications were not clean, and contained other membranous components in the cytoplasm. We tried centrifugation-centric approaches, as well as IP-ing against a mitochondrially-localized tag, but in all cases, the mitochondrial preparations contained other cytoplasmic components. Therefore, we did not feel that this approach was an adequate way to test effects of specifically the mitochondria. We certainly wanted to discuss this aspect in the manuscript, but unfortunately, we were limited due to space. If folks have suggestions on how to best purify mitochondria, we’d love to know, so please reach out.

However, in terms of the bigger question of whether the induced proliferation in cancer cells is specifically due to ROS accumulation in transferred macrophage mitochondria, we tried to address this question with the mito-KillerRed experiments, where we generate ROS using optogenetics, and ask whether this accumulation is sufficient to induce cancer cell proliferation (which we showed it was). We also showed that this same approach could induce Erk activity, and then in separate experiments, we show that macrophage mitochondrial transfer results in accumulation of ROS and increased Erk activity. We feel that these experiments support our conclusions, however, we’d love for a way to link it all together. Unfortunately, we are not convinced that such experiments are possible at this time.

Major Discussion Point #2: Most of the positive examples of transferred mitochondria discussed appeared in a small clump. However, there also appears to be another population that was more diffuse and co-localizes with host mitochondria (e.g., Fig2B, bottom right panels). It would be helpful to show results of these sibling mitochondria for assays performed on their clumpy siblings. If they behave differently, it would be helpful to provide some explanation.

Specific Comment: Figure 2 Majority (57%) of donated mitochondria do not colocalize with LysoTracker signal (N=24 cells, 4 donors) - Here the paper implies that some transferred mitochondria do co-localize with lysoTracker signal. More importantly, they co-localize with host mitochondria. It raises the question of whether they signal through ROS and ERK like their clumpy siblings who are in the limelight of most figures.

🡪Yes, you are correct. There does appear to be a diffuse population of macrophage mitochondria. The majority of these mitochondria co-localize with lysotracker, suggesting that they are being actively degraded. We can’t say that they tend to co-localize with endogenous cancer cell mitochondria, however, it’s possible that this diffuse population is comprised of both mitochondria that are being degraded and mitochondria that are fusing with the endogenous network. We do not know if this population has a different effect on cancer cell behavior because we did not follow this population (mostly because once the mitochondria are degraded or fuse with the network, we can no longer follow those mitochondria!). However, we did follow cancer cells that contained punctate macrophage mitochondria. Often times this was the only population we could observe in the cell at that time, and this is the population in which we observe accumulated ROS.

Major Discussion Point #3:The effects that are attributed to the transferred mitochondria are highly variable (figures 1F, 3A,E) and often due to a subpopulation of samples that show a few extreme values (e.g. figures 2D, 3E, S4B, S4D). This might be expected from effects that are caused by a single mitochondria (which has a small volume) that is transferred to a complete cell. This complicates the study of the transfer process and effects and should be discussed. Also, do the authors have ideas how to improve the system, to make it more robust and easier to study the effects?

🡪The variability in the assays likely reflects the heterogeneity within the biology - Each experiment contains macrophages derived from primary monocytes that are harvested from different human blood donors! Due to the primary nature of these cells, we do expect a range of phenotypes as each donor would have a different genetic background and the monocytes were likely exposed to different environmental stimuli. In fact, even though working on this study was a giant pain due to the variability, we felt more confident about our findings because despite the heterogeneity in the system, we still observed consistent phenotypes. Below we indicate where we took a sample set and removed “outliers”, and ran the statistical tests again. The differences were still statistically significantly different, further suggesting robustness of our findings.

However, we are always on the lookout for ways to make the system easier to study. One way that we will follow up on is using M2-like macrophages since they transfer mitochondria at a higher rate than unstimulated macrophages.

Major Discussion Point #4: The authors conclude that the transfer of dysfunctional mitochondria generated a signal mediated by ROS that activates cell proliferation signals. The statement that "transferred mitochondria act as a signaling source that promotes cancer cell proliferation" is too strong. There is increased ROS production from mitochondria, yes, but an experiment in which ROS are decreased would be needed to properly sustain that conclusion. The title and abstract could be changed to better reflect the data.

Specific Comment: ‘Furthermore, treatment with an ERK inhibitor (ERKi) was sufficient to inhibit ERK activity ‘- curious as to whether antioxidant treatment would reverse any proliferative phenotypes?

🡪We wish we could quench the ROS at macrophage mitochondria. We really tried. We used a combination of ROS quenchers (NAC, mitoTempo, Tempo) and ROS readouts (mitoSOX, CellRox, DCFDA, and the 2 biosensors used in our study: Grx and Orp1), and treated cells for various amounts of time, and no matter what we tried, we could not reliably detect reduction of ROS levels in the host network or the transferred mitochondria (without killing the cells, that is). Another issue that we faced was that any pharmacological treatment would have a global effect on the mitochondrial network in the recipient cells and therefore it would not be possible to distinguish effects from global inhibition of ROS versus specifically at the site of the transferred mitochondria, and we certainly observed cell death upon treatment of ROS quenchers because of this fact. We talked to a couple of ROS experts, and they indicated that this issue is not unique to us, although we unfortunately did not have viable solutions, so if people have ideas or suggestions, please let us know!!

However, despite our failed attempts at quenching ROS, the comment that "transferred mitochondria act as a signaling source that promotes cancer cell proliferation" is too strong of a statement… well, we don’t entirely agree given that we do perform sufficiency experiments in which weinduce ROS and observe both proliferation and ERK signaling, so we do feel reasonably justified to provide the title that we did. However, we will continue to mull over this comment. Thanks for sharing your thoughts.

Major Discussion Point #5:The study may benefit from more direct evidence to support its conclusion of increased proliferation after mitochondrial transfer. While the RNA-seq, flow cytometry, counting of completion of cytokinesis and dry mass measurements provided in the present study do lend some support to the proliferation hypothesis, they all seem indirect. With the biomarkers labeling the mitochondria of donor and potential recipient cells, high content imaging and tracking of cells could be used to monitor cell division. A comparison of cell division rates of transfer-positive cells and transfer-negative cells will provide a more pertinent test of whether mitochondrial transfer promotes recipient cell proliferation.

🡪We should probably do a better job at describing the dry mass measurements (QPI, quantitative phase imaging) because we view this quantification as one of the most direct measurement to monitor cell growth/division. The approach measures the changes in dry mass as the cells prepares for cell division. So not only do we get the final readout of division (complete cytokinesis), but we also get a measure of that growth rate (the cell getting ready to divide) before cytokinesis. This is why we are so tickled to collaborate with Tom Zangle’s lab because we could finally get a direct proliferation readout in real-time. We could also use this approach to follow thousands of cells at a time, a very critical aspect since mitochondrial transfer is rare event, and therefore, we need to follow many cells to have enough statistical power to quantify the growth rates. Check out some of the Zangle lab’s other papers (PMC5866559; PMC6917840; PMC4274116), and please let us know if you disagree with us!

Major Discussion Point #6: The authors have used such a tracking-based approach on a very small scale (n=5) to measure daughter cell growth rate. However, the data do not show a statistically significant difference between the growth rates of daughters that inherited transferred mitochondria and those who did not (Fig S3). Increasing the case number via high content imaging would help obtain sufficient data points for a reliable statistical test. In addition, as suggested above, an accounting of the daughter cells' division rate for transfer positive and negative cells would provide another line of evidence to either prove or disprove the increased proliferation rate hypothesis. The same suggestion goes to the optically induced ERK activation experiments shown in Fig3F. It is also helpful to include references that studied how ERK signaling promotes proliferation and compare the evidence here with evidence or assays used in those studies as a benchmark.

Specific Comment:Figure S3 - There is no statistical test to check for ‘increase in their rate of change of dry mass over time versus sister cells that did not inherit macrophage mitochondria’. What are the colours indicative of in S3B? Can this be reported in the figure legend.

🡪You are right – the tracking-based approach on daughter cells is based on a small ‘n’. However, the tracking itself is performed on 1000s of cells. It’s just that in order to capture daughter cell data, we have to find a cancer cell with macrophage mitochondria (which is only ~1% of the population), and then follow that cell until it divides, and then follow BOTH daughter cells. So, even with the 1000s of cells that we followed, we could only capture a small number of daughter cells. The colors in S3B represent each individual triads – parent and 2 daughters. We will make this info clearer in the legend.

In terms of the optically-induced ERK activation experiments, yes, it would be great to have a higher sampling. These experiments were performed at 63x so we could reliably draw small ROIs to mimic the size of a macrophage mitochondria. While we switched to lower magnification to follow cell division, we still were limited to only a few cells for the actual photoactivation. The technical aspects of this experiment were the reason for the low sampling. Despite these limitations though, we still observed increased cell division upon mito-killerred photoactivation, which we were honestly pretty surprised (and stoked) about.

Other specific comments:

-Figure S1A - The authors could perhaps use a more aggressive gating strategy here, clipping closer to the 231 population described in Fig S1A - picking only the center of the cluster in the upper left of the RFP vs CD11b plot would likely not affect results but make them more convincing by unequivocally excluding macrophages.

-Figure 1D - Not sure about the 0.2% baseline assigned for the monoculture of cancer cells (that does not have the macrophages with the Emerald mitochondria). It is determined with cytometry - I am no expert on that topic, so maybe I missed something - but it looks weird to see some cells with transfer when there is a monoculture.

🡪Due to the variable nature of the mito-mEm signal in the recipient cancer cells (i.e. transfer of one mitochondrion vs transfer of three), we found that an overlap of 0.2% set on a fully stained monoculture control was the most accurate way to gate for the recipient cancer cells. The final gating strategies used in our study were determined by FACS-isolating populations of interest based on several different gating strategies, and directly visualizing cancer cells with macrophage mitochondria without capturing macrophages or cancer cell/macrophage fusions (which is cool, but not what we wanted). To further clarify, there is no transfer occurring in the monoculture – the overlap of mEmerald signal into the transfer gate in that control sample is likely reflective of normally occurring autofluorescence. This is a very important point, so we will make this aspect clearer in the Methods section.

-Figure S1B - Could perhaps be an interesting follow-up question for future works re: differences between cell lines and propensities to transfer mitochondria. Did the authors attempt to use other cell lines (ie, MDCK, HeLa, iPSCs, etc)?

🡪Great question and something that we have also been thinking about. To date the only recipient cells we have used are 231, MCF10A, and PDxO cells. This would be a great avenue for future studies.

-Figure S1B - Did the authors see an increase in growth rate in MCF10A line despite the lower growth rate?

🡪We have not measured the growth rate in MCF10a recipient cells but something that would be great to follow up on in future studies.

-‘physically separated from macrophages by a 0.4μM trans-well insert’ - should this read 0.4 micrometer?

🡪Yes, great catch.

-Figure S1F - The authors wrote that they used a two-way ANOVA analysis, could you report the factors used for that analysis in the Figure legend.

🡪Noted!

-Figure 1B - It is difficult to see the arrowheads in 1B, suggest moving them so they are not covering the magenta fluorescence, have them point from a different angle, and make them more brightly colored. Insets here would help the reader. A negative control image from a monoculture would also be helpful, to ensure the GFP signal is not an artifact of culture conditions.

🡪Thank you for your feedback – we will take note of this.

-Figure 1F - For graphs that do not show zero (as in 1F), the bar should be omitted. In these cases the length of the bar does not reflect the average of the data (as it does in 1D).

-Figure 3C - Please omit bar, see comment on panel 1F.

🡪 In the case of Fig 1F, we modified the y-axis to eliminate empty space. The bar is representative of mean of the data displayed in both 1D as well as 1F, but we can add a broken y-axis to help make this point.

-Figure 1 - Given that these data are fractions of a population (ie. can be described via a contingency table), isn't something like a Fisher's exact test a better measure of significance here?

🡪We think you are referring to Figure 1D? If so, we thought that we could not use Fisher’s exact test because that test assumed parametric distributions (which we do not observe). We have been working with a biostatistician for our statistics, but please do let us know if we have it wrong.

-Single cell RNA- sequencing - In the methods section the authors mention doing a differential analysis between the cells that received the mitochondria and the cells that didn’t. It might be worth introducing a figure (a heatmap or a U-MAP) relating to this analysis. Single cell sequencing would not only affirm the heterogeneity between these two populations but also help in highlighting the novel cell surface markers associated with the two populations.

🡪Yeah, good point – we can add a UMAP.

-‘mito-mEm+ mitochondria remained distinct from the recipient host mitochondrial network, with no detectable loss of the fluorescent signal for over 15 hours’- It is surprising that the transferred mitochondria do (or cannot) fuse with the host 231 mitochondria.

🡪We were also initially surprised to find that the transferred mitochondria do not fuse with the host 231 network! We think that the lack of fusion is due to the fact that the transferred mitochondria do not exhibit membrane potential (which is required for mitochondrial fusion). We also think that these results open interesting lines of questioning: Why are these depolarized mitochondria not degraded? Is this an active avoidance of the mitophagy pathway? How dynamic are these punctae? Many fun and interesting questions regarding the long-lived nature of these transferred mitochondria.

-It is unclear in these images, but the 231 mitochondria appear fragmented too. Is it possible that the mitochondrial fusion machinery (Opa1 or Mfn1/2) are inactive?

🡪231 cells are capable of fission and fusion (PMC7275541, PMC3911914, and in our own timelapse recordings), so we think that the machinery is functional. However, we don’t know whether the 231 mitochondrial machinery changes after receipt of macrophage mitochondria. Interestingly, the references above both investigate how mitochondrial dynamics promote tumor metastasis. A fascinating future direction could include an investigation to how macrophage mitochondrial transfer influences tumor cell mitochondrial dynamics.

-Figure 2B - What does the MTDR staining of the macrophage mitochondria prior to transfer look like? Important to check this to confirm that only the transferred mitochondria had lower membrane potential.

-‘significantly higher ratios of oxidized:reduced protein were associated with the transferred mitochondria versus the host network’-Here too, it would be important to check the mito-Grx1-roGFP2 readout of macrophage mitochondria prior to transfer.

🡪The way that these comments are written is as if we already know that the mitochondria are dysfunctionalbefore transfer to cancer cells. But we actually do not know if that is the case. It’s also possible that macrophage mitochondria become dysfunctional once they are in the cancer cell, which would be equally cool. So, we are actively investigating this biology.

-Figure 2A, 2BB and S1D - How were the colocalizations assessed? Was it just a visual assessment? Given the importance of these experiments for the whole story, having a quantification of the level of colocalization with each dye would be important.

🡪This is a good point and it should be straightforward to include a Pearsons coefficient for these markers.

-Figure S1D - The paper makes an argument about mitochondria transferred from Macrophages (marked green) having positive DNA stain (gray), but appearing depolarized (negative TMRM stain). The image in FigS1D is peculiar, as the majority of the 231 cells' mitochondria appear to not have any DNA stain but maintain membrane potential (positive in TMRM), while some (just above the green macrophage mitochondria) do have both DNA stain and membrane potential. The authors might want to clarify whether this is a typical scenario, and if so perhaps offer an explanation as to why the 231 mitochondria exhibit such heterogeneity.

🡪The images in S1D are of a single z-plane image therefore the DNA signal in the endogenous network is more readily visible in planes that are not shown.

-‘we confirmed that 91% of transferred mitochondria were not encapsulated by a membranous structure, thus excluding sequestration as a mechanism for explaining the lack of degradation or interaction with the endogenous mitochondrial network’ - This is based on co-staining with MemBrite 640/660, which is a dye that "covalently labels the surface of live cells", thus there is a concern as to whether this approach allows to study whether the mitochondrium is encapsulated by an endomembrane.

🡪Thank you for your feedback. We actually do think that Membrite can label endomembrane in addition to the plasma membrane. This is from the published Membrite protocol: “MemBrite™ Fix dyes are designed to be fixed shortly after staining, when they primarily localize to the plasma membrane/cell surface. Cells also can be returned to growth medium and cultured after staining, however, dye localization in live cells changes over time. Labeled membranes become internalized, so staining gradually changes from cell surface to intracellular vesicles, usually becoming mostly intracellular after about 24 hours. Internalized MemBrite™ Fix dye is usually detectable for up to 48 hours after staining, though this may vary by cell type”.

In our hands, we found that the dye started to become internalized and labeled vesicles within the cell within a few hours of staining. The images in the panels that you refer to came from time-lapse imaging experiments of between 10-15 hours, therefore the cells have internalized the MemBrite signal allowing for the visualization of internal vesicles. Also, in other studies not in the preprint, we perfused purified mitochondrial preparations onto 231 cells. The 231 cells took up the mitochondria from the environment, and all of these engulfed mitochondria were surrounded by a MemBrite positive membrane! These results further suggest that if the transferred mitochondria were encapsulated by a membrane, we would be able to visualize it.

_-‘macrophage mitochondria are depolarized but remain in the recipient cancer cell’ -_Did the authors examine the extent of cancer cell death in their co-culture system (due to the activation of apoptosis by the depolarized mitochondria)?

🡪We do not find any evidence of abnormal levels of cell death by both flow cytometry assays as well through our QPI image analysis.

-Figure 2C–D - Like in Fig 2B, in the bottom left of panel of Fig 2C there are a lot of donor mitochondria not in highly oxidized state and the growth/proliferation phenotypes apply mostly to donor mitochondria that appear 'clumpy'.

-Perhaps it is worth commenting on whether there is a link between donor mitochondrial morphology and the suspected proliferation-enhancing phenotype.

🡪The images in Fig. 2C are of the same cell – a single recipient cancer cell which is expressing the Grx biosensor. The donor mitochondria are labeled with an arrowhead, the rest of the yellow/green signal (bottom right) is from the endogenous host network and therefore we do not expect it to be in a highly oxidized state (ie. more yellow than green).

Regarding the mito morphology and proliferation – great question, and one that we are actively working on!

-‘At 24 hours, we observed a similar trend, but no statistically significant difference (Fig. S4D). These results indicate ROS accumulates at the site of transferred mitochondria in recipient cancer cells’ - if a specific sensor fails to show a significant oxidation at 24 hours compared mito-Grx1-roGFP2 which reports on mitochondrial glutathione redox state, does that mean there are ROS independent ways to oxidize Glutathione? The authors did see cell growth phenotype both in 24 and 48 hours which suggests that something is happening in 24 hours despite no significant difference in ROS H2O2 sensor.

🡪The additional biosensor that we used – mito-Orp1-roGFP2 - has been engineered to be a readout of one type of ROS – H2O2. The Grx probe is a surrogate for ROS of any type, of which there are many! To us, it is not completely unexpected that they would behave differently over time since they are readout for two separate things, and it generates an interesting possibility that different types of ROS accumulate over time. Given that the Grx probe shows an increase at 24 hours, which is when we observe the proliferation phenotype, we think we are on the right track. If you have ideas on robust ways to directly observe specific types of ROS, we would love to know!

-The differences in ratio for the two sensors used are not very convincing. In Fig 2D and Fig S4B and D the “host” and “transfer” populations are very similar. The difference seems only due to the presence of a few outliers in the “transfer” populations. More importantly, sometimes it seems that these outliers come mostly from one donor rather than being present in all 3 donors. It could be good to show histograms of the two populations for each replicate/donor and maybe redo the stats excluding these outliers.

🡪We think that the heterogeneity that is observed is due to the biology in the system – we are using primary macrophages derived from blood donors. However, for the data represented in Fig 2D, just as a test case, we took out the top four “outliers” in that data set and re-ran the Wilcoxon matched-pairs signed rank test and the p-value was 0.0010 (***), further suggesting that the ROS biosensors are revealing consistent and robust results.

-Figure S5C - it seems like the percentage of cells that divided is the same for unstimulated cells and cells with stimulated mito-KillerRed. Isn't this contrary to the expectation? The figure shows that photobleaching cytoplasm decreased % cell division, which is puzzling.

🡪The mean percent of cells that divided in unstimulated and mito bleach are very similar and was not significantly different. One point to be made that may not be well illustrated in our graphical representation is that if you look at the matched data (points connected are averaged per FOV for each condition in the same experiment) the trend shows that the mito bleach does seem to have an increase in cell division which is washed out with the average bar overlay. We should note that this experiment is very “noisy” and therefore we needed a lot of N to be able to detect significant changes. We are currently thinking about other ways to demonstrate sufficiency as it relates to cell proliferation – any experimental suggestions would be very welcome! Thanks for the feedback.

-Figure 3A - In the 'cyto' condition 6 out of 13 fields have no cells that divide. Is that expected? What is the percentage of dividing cells for cells that were not illuminated at all (a control that is lacking)? There is large variation, ranging from 0% to 22%. The evidence that illumination of KillerRed leads to increased proliferation is rather weak. Also, since Cyto and Mito are different cells, is a "paired" statistical test the right kind of test to use here?

🡪Additional data pertaining to Fig. 3A can be found in Fig. S5C, which includes the control for cells not illuminated at all. Having no cells that divide in a field of view is not surprising to us – the doubling time for these cells is ~35 hours, and we imaged for 18 hours. Also, for each field of view, our ‘n’ for each field of view was often 6-8 cells because we performed these experiments at 63X to allow for accurately drawn regions of interest for photoactivation. We also internally controlled every experiment (each experiment consisted of fields of view that had either mito activation, cyto activation, or no-activation controls, all of which were imaged overnight with multiple x/y positions). Cells that left the field of view over the 18 hours of imaging could not be quantified. It’s this sampling that caused the large variation in the graph. But again, as with many of our experiments, despite this variability, we still observe a significant difference in our experimental conditions over control cyto bleach. As for the statistical test, our understanding is that given each experiment is internally controlled, and we compare within each experiment, a paired statistical test is appropriate here. We will consult with our biostatistician to confirm, though.

-‘ROS induces several downstream signaling pathways’ - We would not expect the authors to investigate every signaling pathway, but wonder if the PI3K pathway was explored? It seems to be the other major cancer/proliferative pathway induced by ROS.

🡪Yes, this is a very good point! We actually assessed three different pathways at first – ERK, PI3K-AKT, and NLRP3/inflammasome. While analyzing these 3 pathways simultaneously, we discovered that ERK inhibitors resulted in decreased proliferation in cancer cells with macrophage mitochondria. As a result, we then focused on the ERK pathway. We still do not know if PI3K-AKT or NLRP3/inflammasome pathways play a role in this biology because we have not gone back and revisited these experiments yet, however in figure 3F, ERKi treated recipient cells exhibit a partial ‘rescue’ of baseline proliferation. This suggests that other pathways may indeed be involved and we plan to investigate this possibility.

-‘Recipient 231 cells had significantly higher cytoplasmic to nuclear (C/N) ERK-KTR ratios compared to cells that did not receive transfer’-Since two different quantification styles with opposite fraction values were used, is it possible to please specify which one was used here.

🡪Will do!

-Figure 3B - Please show the outlines of the nuclei and that of the cell.

🡪That would be helpful, wouldn’t it? Will do!

-Figure 3D - it is peculiar that ERK-KTR in Fig 3D is so strongly cytosolic while in Fig 3B it is almost exclusively nuclear. If this sensor behaves differently in different situations, the authors may want to comment on how that would affect their conclusions.

🡪The panels in Fig. 3B were taken with the ImageStream flow cytometer which takes a lower resolution image of a single plane of a cell in suspension in the flow stream. In Fig. 3D, those images are from confocal spinning disk microscopy which allows for higher resolution, z-stack images of adherent cells on glass. Therefore, we think the differences that you point out are likely due to the fact that the two images come from very different imaging systems.

-Figure 3E - The effect of 'opto-induced' ERK activity is weak. The initial ERK-KTR is 1 at time point zero (as the data is normalized to this timepoint) and around 1 for both the cyto and mito condition. A statistical difference is observed, but the effect is minor and it is unclear whether it is biologically meaningful. The 'cyto' condition shows an average below 1 and the mito condition remains 1, suggesting that ERK activity remains constant when ROS are produced in the mitochondria.

-Also from S8C and 3E it appears cyto actually shows a decrease rather than mito showing an increase, could the authors comment on this?

🡪We have a few thoughts on this. The first is that we don’t expect a dramatic change in ERK signaling because the ROS accumulation is localized to a small region in the recipient cell. This is not a situation where we would expect a large-scale change because we are adding a growth factor. We can understand that the change in ERK activity may appear to be minor, but our data suggest that these subtle changes in kinase signaling translate into significant changes in downstream behavior – proliferation. The way that we interpret differences as “biological meaningful” is whether they exhibit a functional response, and in our study, we show that inhibiting the induction of ERK activity in cancer cells with macrophage mitos inhibits proliferation. What is most interesting to us is that cancer cells that do not have macrophage mitochondria have an unchanged fraction of cells in G2/M phase of the cell cycle in response to the concentration of ERK inhibitor we used, suggesting that the ERK inhibition specifically blocks macrophage mitochondria-induced proliferation.

In Fig. S8C, bleaching a region of cytoplasm does seem to cause a decrease in ERK activity over time. We really can’t explain this result. However, we do think that ERK activity is higher in mito-bleached cells because mt-ROS is generating an increase in ERK activity which compensates for the decrease in activity that occurs when the cytoplasmic region of interest is photobleached. It’s still a head scratcher, though, but we did perform internal controls for every experiment (as we describe above), and the mito-bleach, cyto-bleach, and no-bleach conditions were run side-by-side such that we can make apples-to-apples comparisons.

-‘patient-derived xenografts (PDxOs)’ - As a control it would be relevant to include a normal mammary organoid model perhaps from the same patient to demonstrate that the transfer of mitochondria specifically to the cancer cells is more beneficial.

🡪Using a normal mammary organoid cells as a control to compare efficiency of transfer and downstream phenotypes would be very interesting. Due to the fact that these are patient-derived organoids, we are unable to acquire non-malignant cells from the same patient. Expanding our studies in the MCF10A cell line that we utilized in this paper would be an alternative to what you propose and would also expand our understanding of general biology underlying mitochondrial transfer.

-‘macrophages to both HCI-037 and HCI-038 PDxO cells (Fig. 4G)’ - Why is M0 able to transfer efficiently to HCL-037 tumour when its mitochondrial network is less fragmented as M2?

🡪These results really stood out to us. It was quite surprising that in HCI-037, both M0 and M2 macrophages were able to transfer their mitochondria at similar efficiencies, but in HCI-038, M2 macrophages were more efficient at transfer. HCI-037 is a primary tumor, and HCI-038 is a metastases from the same patient, so there are some exciting avenues of study to examine how macrophage mitochondria transfer differs at the primary versus metastatic site. There is still very little known about how donor cell dynamics influence mitochondrial transfer!

-Are mito transfer from M0 depolarised and accumulate ROS or show increased ERK activity or increased cell proliferation?

🡪Yes – all studies, except studies pertinent to fig 4 (where we assessed macrophage differentiation states), were done with M0 macrophages.

-‘M2-like macrophages preferentially transferred mitochondria to the bone metastasis PDxO cells (HCI-038) when compared to primary breast tumor PDxO cells (HCI-037)’ -The authors may want to check this statement here as it is in consistent with their data plot. In Fig. 4G, M2/PDxO transfer percentages for HCI-037 and HCI-038 are about the same, unless the authors provide statistical tests to prove otherwise. Instead, M0 appears to transfer mitochondria to HCI-037 much more efficiently than it does HCI-038.

🡪Upon re-reading our sentence again, we now realize that it’s actually quite poorly written, so we can understand the confusion! What we meant to articulate is that M2-like macrophages are better at transferring mitochondria to HCI-038 than M0 macrophages. Whereas in HCI-037, we do not observe the same preferential transfer (ie. M0 and M2 can transfer at the same efficiency). We will certainly clarify this language in the manuscript.

-‘M2-like macrophages exhibit mitochondrial fragmentation’ - Is there a correlation between the status of the mitochondrial network in the donor and the % of transfer to the recipient? If so, this would be a correlation that would support the conclusions.

🡪Yes, please see Fig. 4C for transfer rates with different donor subtypes and Fig. 4H for a general working model on how we think these data fit into the larger picture.

-‘accumulate ROS, leading to increased ERK activity’ - Did the authors obtain similar results with the PDXOs? It would be an interesting observation if the primary samples also exhibit a mechanism similar to established cell lines wherein there are more accumulated genetic changes.

🡪Our main limitation with PDxOs is overcoming the technical hurdles related to our downstream assays. These include introducing relevant reporters and generating stable lines in the PDxOs, and imaging at high-resolution when the PDxOs are cultured in 3D. However, we are very interested in this question as well, and are actively thinking about ways to overcome these hurdles.

-It would also be interesting to examine whether there is any difference in the ROS-ERK mechanism for primary and metastatic tumour.

🡪We agree and this is an active avenue of investigation for us. We agree and are currently pursing models to understand how our findings fit into the larger picture of tumorigenesis and metastatic potential. We had spent months pursuing anin vivo approach using a murine Cre/LoxP system to genetically label mouse macrophage mitochondria with GFP. We crossed mice which express Cre under a monocyte-specific promoter (Jax, SN: 004781) and mice with germline expression of Lox-Stop-Lox-3xHA-EGFP-OMP25 (Jax, SN: 032290) with the expectation of seeing Cre-based excision of the stop cassette – thus resulting in offspring with macrophages expressing mitochondrial-localized GFP. However, the macrophages of the resulting offspring do not express GFP (by flow cytometry, imaging, and western blot analysis), despite the PCR-verified presence of both transgenes and the excision of the stop cassette. Needless to say, this was quite frustrating! We are currently in the process of developing a newly available MitoTag model which has been optimized for visualization purposes (Jax, SN: 032675). If you have any suggestions or advice on this matter we would much appreciate your thoughts!

-‘in cancer cells that receive exogenous mitochondria’ - Since these macrophages also transfer mitochondria to non-malignant cells, such as MCF10A cells shown in Fig S1B, perhaps the authors could comment on whether this is part of a physiological process that would also promote normal cell growth?

🡪 There are so many questions regarding when and why macrophages might transfer mitochondria. In general, mitochondrial transfer is observed in stressed cells. Our data suggest that transfer happens to MCF10A cells although at a much lower rate than their malignant counterparts, 231 cells, but we do not know whether similar downstream mechanisms and phenotypes are also occurring in the non-malignant cells. Thanks for your feedback – more to come here!

DOI: 10.1016/j.scr.2021.102520

Resource: (Abcam Cat# ab9110, RRID:AB_307019)

Curator: @Naa003

SciCrunch record: RRID:AB_307019

Curator comments: Rabbit Anti-HA tag Polyclonal Antibody, Unconjugated Abcam Cat# ab9110

---

What is this?

Good question. Too bad it went unanswered.

headings to tag / organize information under generally. Refining / honing tags with further descriptors would be ideal.

i.e.: Spiritual / Why i.e.: INTELLECTUAL / MEDICINE / PRACTICE / INTEGUMENTARY

second tag

Reviewer #1 (Public Review):

In the present technical report the authors develop a novel and simple strategy to generate and efficiently isolate cre/lox responsive alleles that in the right orientation are highly mutagenic. These allele offer in principle a powerful tool for the tissue specific analysis of gene function giving the ability to inactivate the gene of interest with high temporal and spatial control. The mutagenic targeting cassette contains a marker reporter for the efficient isolation of putative carriers following the integration in injected zebrafish embryos. The cassette in the mutagenic orientation also potentially labels the disrupted gene with a fluorescent tag, with the intent of following the single cells (and their full lineage) in which gene disruption has occurred. This approach is applied to three test loci comparing for which the same group had generated classical null alleles containing small deletions in the coding region.

SciScore for 10.1101/2021.09.03.458953: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein and peptide production: The S. pombe αCOPI-WD40 domain was synthesized by TOPGENE and cloned in pcDNA3.1(+) with a C-terminal strep-tag for affinity purification.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1(+)</div><div>suggested: RRID:Addgene_129020)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">β’COPI-WD40 (residues 1-304) from Saccharomyces cerevisiae was cloned in pGEX-6P-1 vector and expressed overnight in E. coli pLysS cells at 18°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX-6P-1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression was performed in Expi293 mammalian cells using the Thermo Fisher ExpiFectamine expression kit.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Thermo Fisher ExpiFectamine</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecular replacement was performed in Phenix using a previously determined αCOPI-WD40 domain structure (PDB ID 4J87) as the search model (McCoy et al., 2007; Rossmann and Blow, 1962).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative model building and refinement were performed in Phenix.refine (Afonine et al., 2012) and Coot (Emsley and Cowtan, 2004).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were generated in PyMol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis of Ramachandran angles: The crystal structures of wild type, Arg57→Ala, and Tyr139→Ala αCOPI-WD40 were analyzed in Molprobity (Williams et al., 2018).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Default parameters were used, with the exception of extra rotamers allowed during packing of modeled side chains, specified by command line parameters: Sequence analysis of dibasic motifs in the human membrane proteome: UNIPROT identifiers of secreted and membrane-bound human proteins, as well as secreted/membrane-bound protein isoforms, were downloaded from the Human Protein Atlas (http://www.proteinatlas.org) (Thul et al., 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>http://www.proteinatlas.org</div><div>suggested: (HPA, RRID:SCR_006710)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your data.

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.09.03.458829: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: This study was approved by the Institution Review Board of Tsinghua University (20210040)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: Cells were tested routinely and found to be free of mycoplasma contamination.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture: HEK293T (American Tissue Culture Collection, ATCC, Manassas, VA, CRL-3216), Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and A549 (ATCC) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, NY, USA) supplemented with 10% (vol/vol</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Surface ACE2 binding with RBD-His assay: HeLa cells were transduced with lentiviruses expressing the ACE2 variants for 48 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of SARS-CoV-2 pseudotyped virus, determination of viral entry efficiency and analysis of spike protein cleavage: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G (Addgene #12259)) using VigoFect (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For neutralization experiments, S protein bearing pseudotyped virion particles were pre-incubated for 30 min at 37°C with diluted plasma samples obtained from convalescent COVID-19 patients, before the mixtures were inoculated onto HeLa-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant ACE2-lg protein expression and purification: ACE2-lg, a recombinant Fc fusion protein of soluble human ACE2 (residues Gln18-Ser740) was expressed in 293F cells and purified using protein A affinity chromatography as described in our previous study28.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293F</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids: The cDNAs encoding the ACE2 orthologs were synthesized by GenScript and cloned into the pLVX-IRES-zsGreen1 vector (Catalog No. 632187, Clontech Laboratories, Inc) with a C-terminal FLAG tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-IRES-zsGreen1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of SARS-CoV-2 pseudotyped virus, determination of viral entry efficiency and analysis of spike protein cleavage: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G (Addgene #12259)) using VigoFect (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTG-MLV-Fluc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pTG-MLV-Gag-pol</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div><div style="margin-bottom:8px"><div>pMD2 . G</div><div>suggested: None</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 25, 26 and 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.09.01.458653: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-FLAG epitope (DYKDDDDK, catalog# 637301) and horseradish peroxidase (HRP) Donkey anti-human IgG antibody (catalog #410902) were purchased from Biolegend (San Diego, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-FLAG epitope (DYKDDDDK, catalog# 637301)</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-FLAG epitope (DYKDDDDK</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (BioLegend Cat# 410902, RRID:AB_2686937)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The secondary antibody anti-rat IgG HRP (catalog# 7077) was bought from Cell Signaling Technology (CST, Danvers, MA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>The secondary antibody anti-rat IgG HRP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rat IgG</div><div>suggested: (Cell Signaling Technology Cat# 7077, RRID:AB_10694715)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293T cells expressing human angiotensin I-converting enzyme 2 (HEK293T-hACE2) were kindly provided by Dr. Jesse Bloom (Fred Hutchinson Cancer Research Center, Seattle, USA) (51).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lenti-X 293T cell line was purchased from Takara Bio USA Inc. (San Jose, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1 hr incubation, the cellulose paper treated pseudovirus was gently transferred to transfect HEK293T-hACE2 cells with 80% confluency, followed by adding polybrene to a final concentration of 8 μg/ml.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-hACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids construction, protein expression and purification: Ty1 variants, including Ty1-CBD and control protein Ty1 without CBD module, were cloned into pSH200 vector (a generous gift from Prof. Xiling Shen at Duke University) containing 6 x Histidine tag (His-tag), between BamHI and XbaI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSH200</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Detergent compatible (DC) protein assay kit was bought from Bio-Rad Laboratories (Hercules, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bio-Rad Laboratories</div><div>suggested: (Bio-Rad Laboratories, RRID:SCR_008426)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After the final wash, the cells were resuspended in 200 μL FACS buffer (5% FBS, 2mM EDTA, 0.1% sodium azide in PBS) for flow cytometric analysis in Attune™ NxT Flow Cytometer (Thermofisher) and data were analyzed by FlowJo (Franklin Lakes, NJ).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical significance was evaluated using one-way ANOVA followed by Tukey post hoc test using GraphPad PRISM (San Diego, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad PRISM</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Despite the promises demonstrated in this study, one limitation is that only pseudovirus-containing culture media were used to characterize the fusion proteins for proof-of-concept. Therefore, it is necessary to further evaluate our approach in real specimens such as blood from COVID-19 patients in the near future.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

It's not everyday I get tagged (explicitly added as a meta-data tag) on someone's website, much less one as well-curated as this one.

I don't know which language tag to use as the text is not in latin letters.

I don't know which language tag to use as the text is not written in latin letters.

Boy, we of the Markdown persuasion sure do have some catching up to do with the outliners where this kind of thing is concerned...

new window => new tab

SciScore for 10.1101/2021.08.28.458041: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protease-Glo luciferase assay was carried out as follows: 293T cells in 10 cm culture dish were transfected with pGlosensor-30F Mpro plasmid in the presence of transfection reagent TransIT-293 (Mirus catalog no. MIR 2700) according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Calu-3 cells (ATCC, HTB-55) were plated in 384 well plates and grown in Minimal Eagles Medium supplemented with 1% non-essential amino acids, 1% penicillin/streptomycin, and 10% FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein Expression and Purification: The tag-free SARS CoV-2 Mpro protein with native N- and C-termini was expressed in pSUMO construct as described previously3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSUMO</div><div>suggested: RRID:Addgene_170732)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">50 ng of FlipGFP-Mpro plasmid and 50 ng SARS CoV-2 Mpro expression plasmid pcDNA3.1 SARSCoV-2 Mpro were transfected into each well with transfection reagent TransIT-293 (Mirus catalog no. MIR 2700) according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlipGFP-Mpro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3.1 SARSCoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protease-Glo luciferase assay: pGlosensor-30F DEVD vector was obtained from Promega (Catlog no. CS182101).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGlosensor-30F DEVD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protease-Glo luciferase assay was carried out as follows: 293T cells in 10 cm culture dish were transfected with pGlosensor-30F Mpro plasmid in the presence of transfection reagent TransIT-293 (Mirus catalog no. MIR 2700) according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGlosensor-30F Mpro</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two days after transfection, images were taken with Cytation 5 imaging reader (Biotek) using GFP and mCherry channels via 10× objective lens and were analyzed with Gen5 3.10 software (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gen5</div><div>suggested: (Gen5, RRID:SCR_017317)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A non-linear regression curve fit analysis (GraphPad Prism 8) of POC Infection and cell viability versus the log10 transformed concentration values to calculate EC50 values for Infection and CC50 values for cell viability.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

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About SciScore

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Author Response:

Reviewer #3:

The authors modified a previously reported hybrid cytochrome bcc-aa3 supercomplex, consisting of bcc from M. tuberculosis and aa3 from M. smegmatis, (Kim et al 2015) by appending an affinity tag facilitating purification. The cryo-EM experiments are based on the authors' earlier work (Gong et al. 2018) on the structure of the bcc-aa3 supercomplex from M. smegmatis. The authors then determine the structure of the bcc part alone and in complex with Q203 and TB47.

The manuscript is well written and the obtained results are presented in a concise, clear-cut manner. In general, the data support the conclusions drawn.

We thank the reviewer for this evaluation.

To this reviewer, the following points are unclear:

1. The purified enzyme elutes from the gel filtration column as one peak, but there seems to be no information given on the subunit composition and the enzymatic activity of the purified hybrid cytochrome bcc-aa3 supercomplex.

See answers to Question 1 from the major Essential Revisions and Question 1 from the minor Essential Revisions.

"We have now shown that the purified chimeric supercomplex is a functional assembly with a (mean ± s.d., n = 4), in agreement with the previous study that shows M. tuberculosis CIII can functionally complement native M. smegmatis CIII and maintain the growth of M. smegmatis (Kim et al., 2015). The in vitro inhibitions of this enzyme by Q203 and TB47 was determined by means of an DMNQH2/oxygen oxidoreductase activity assay. In the assay, 500 nM Q203 or TB47 was chosen, which is close to the median inhibitory concentration (IC50) obtained from the menadiol-induced oxygen consumption in our previous study (Gong et al., 2018). After addition of Q203 and TB47, the values of turnover number of the hybrid supercomplex are reduced to 5.8 +/- 2.4 e-s-1 (Figure 4-figure supplement 4) and 5.1 +/- 2.9 e-s-1 (Figure 5-figure supplement 4) respectively, from 23.3 +/- 2.4 e-s-1. We have incorporated this new data into the text (lines 90-93, 187-189, 206-209)."

"The subunit composition of the purified enzyme has now been provided in Figure 2-figure supplement 1."

1. It is unclear what is the conclusion of the structure comparison (Fig 6) is regarding the affinity of Q203 for M. smegmatis.

The structural comparison indicates that Q203 should have a similar binding mechanism and a similar effect on the activity of cytochrome bcc from M. smegmatis and M. tuberculosis. This is in good agreement with previous antimycobacterial activity data and inhibition data for the bcc complexes from M. smegmatis and M. tuberculosis (Gong et al., 2018; Lu et al., 2018a). These have now been incorporated into the revised manuscript (line 223-227).

SciScore for 10.1101/2021.08.27.457964: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The N protein sequence (GenBank QIG56001.1) was amplified from cDNA samples using primers SC2-protN28182-F (5’-AGTCTTGTAGTGCGTTGTTCG-3’) and SC2-protN29566-R (5’-ATAGCCCATCTGCCTTGTGT-3’) and cloned into pGEM-T Easy (PROMEGA - USA), generating plasmid pGEM-SC2-N.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEM-T Easy</div><div>suggested: RRID:Addgene_80557)</div></div><div style="margin-bottom:8px"><div>pGEM-SC2-N</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The N sequence was reamplified from pGEM-SC2N with forward 5’-AACAAGCTAGCATGTCTGATAATGGACCCCAAAATCAG-3’ and reverse 5’-GGTCTGCGGCCGCTTAGGCCTGAGTTGAGTCAGCACTGCT-3’ primers and subcloned into the NheI/NotI sites of a pET28a-TEV vector carrying a 6xHis-tag and TEV protease cleavage site at the N-terminus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEM-SC2N</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pET28a-TEV</div><div>suggested: RRID:Addgene_168267)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, the initial 3D map was refined in Imagic using an iterative process of angular reconstitution and class average rotation and translation alignment to 3D reprojections, achieving 3D resolution convergence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Imagic</div><div>suggested: (IMAGIC, RRID:SCR_014447)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CG molecular dynamics simulations: CG molecular dynamics simulations were performed using CafeMol 3.1 software52.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CafeMol</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">An initial N protein dimer all-atom model was built in YASARA software53 using crystallographic structures of NTD monomer (PDB ID: 6VYO) and CTD dimer (PDB ID: 6WJI).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>YASARA</div><div>suggested: (YASARA, RRID:SCR_017591)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Tools for comparing CG simulations with experimental data: For comparing CG simulation radius of gyration with experimental data, we estimated the radius of gyration of the simulated systems with Bio3D package using an in-house R script54.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bio3D</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The area of 2D reprojections was determined using ImageJ software56. 3D Atomic model fitting: From the previous independent simulation of the CG model of N protein dimer in complex with a 60-nt-long RNA were selected 100 structures with the lowest radius of gyration using only the structures domains (NTDs and CTDs).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The molecular interactions were described by CHARMM36 force field with CMAP corrections60.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CMAP</div><div>suggested: (CMAP, RRID:SCR_009034)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All simulations were performed with NAMD 2.1361.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NAMD</div><div>suggested: (NAMD, RRID:SCR_014894)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the systems were described using Amber ff14SB force field68 and the topologies were generated using tLeap program from AmberTool2069.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Amber</div><div>suggested: (AMBER, RRID:SCR_016151)</div></div><div style="margin-bottom:8px"><div>tLeap</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The images of the C-terminal tail monomers and dimers were generated using pymol 2.3.0 (open-source build).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pymol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 38. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Numerical identification unique to each student for purposes for/relating to services, processes, concepts in specific or multiple collegial institutions

I.E. Personal Identification Tag

This would allow us to add a tag to our annotation which is related to what we are annotating, in order to make it easier to find annotations by specifics.

SciScore for 10.1101/2021.08.25.457644: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Euthanasia Agents: On day 4 post challenge half of the animals per group were euthanized by exsanguination under isoflurane anesthesia and necropsy was performed, with the remaining half of the animals following on day 7 post challenge.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Vaccinations: BALB/c mice (OlaHSD; Envigo, female, 8-9 weeks old at day 0) were immunized on day 0 and 21 via the intranasal or intramuscular route.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">On day 0, 21 and 35, blood was collected for assessment of induction antibodies against spike and SARS-CoV-2 specific neutralizing antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 specific neutralizing antibodies .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">On day 35 nasal washes and the lungs were also collected for IgA antibody determination.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing another three times the HRP conjugated antibody (Goat-anti-mouse HRP IgG 1:8000, IgG1 1:4000</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HRP IgG</div><div>suggested: (Bioss Cat# bs-4000R-HRP, RRID:AB_11081943)</div></div><div style="margin-bottom:8px"><div>IgG1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, the virus-antibody mixtures are transferred to plates with Vero E6 cell culture monolayers, followed by an incubation period of 5-6 days at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vaccinations: BALB/c mice (OlaHSD; Envigo, female, 8-9 weeks old at day 0) were immunized on day 0 and 21 via the intranasal or intramuscular route.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

A significant limitation for the use of viral vector-based vaccines is the development of anti-vector immunity, which restricts the number of possible booster vaccinations. With OMV based vaccines we assume this is less of an issue, as they are non-replicating and do not depend on a specific host receptor for cellular entry. In addition, major antigens can be removed by gene deletion, as we did with the immunodominant PorA outer membrane protein in our vaccine strain, thus limiting the response against neisserial antigens. OMV-spike vaccination might thus also find an application as a heterologous boost after primary vaccination with viral vector-based vaccines. Overall, here we show that OMV-mC-Spike is safe and effective in both mice and hamsters. In these animal models, intranasal vaccination with OMV-mC-Spike is superior to intramuscular vaccination, since the amount of IgG induced is higher and in addition a strong mucosal response is induced. Our study demonstrates how adding an mCRAMP tag to the Spike protein and combining it with meningococcal OMVs improves its immunogenicity, thus warranting further development of this vaccine concept towards clinical trials in humans.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.08.25.457627: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Animal care and ethics statement: All animal experiments were conducted in animal biosafety level 3 (BSL3) facilities at the Biosecurity Research Institute at Kansas State University according to protocols approved by the Institutional Animal Care and Use Committee at Kansas State University and the guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 antibodies from humans, cats, and rabbits: Convalescent sera (Lotus 11 and 25) from COVID-19 patients were obtained from Dr. Thomas Rogers from the Scripps Research Institute, San Diego, CA, USA. Cat sera (Cat 247 and 903) were collected from cats enrolled in SARS-CoV-2 re-infection studies [89]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of each species’ ACE2 receptor in the cells was confirmed by Western Blot analysis using antibody to human ACE2 (Abcam, Waltham, MA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human ACE2</div><div>suggested: (Abcam Cat# 3149-1, RRID:AB_2242331)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and plasmids: Human embryonic kidney 293 (HEK293), the Crandell-Rees feline kidney (CRFK) and Calu-3 cells were purchased from American Type Culture Collection (ATCC; Manassas, VA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: BCRJ Cat# 0264, RRID:CVCL_0609)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 cells expressing human TMPRSS2 (Vero-TMPRSS2) cells were obtained from Creative Biogene (Shirley, NY)[88].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation of SARS-CoV-2 S pseudotyped viruses: The 2nd generation lentiviral packaging plasmid, psPAX2 (Addgene), a reporter plasmid pUCGFP-Luc (Addgene), and parental or mutant pAbVec-SARS2-S were transfected into HEK293 cells to produce pseudotyped viruses.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SA/KRISP-K005325/2020 stock was subsequently passaged on Calu3 cells and NGS results showed this stock to contain only 13% of the furin site mutation; this stock was used for the in vitro virus replication kinetic experiments.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Inoculum (defined as 0 hpi) and the time point collected supernatants were then titrated on Vero-TMPRSS2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nasal wash and lung homogenates were filtered through a 0.2 μm filter prior to virus titration on Vero E6-TMPRSS2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The codon-optimized cDNAs of the open reading frame of the human or animal ACE2 gene with FLAG tag were synthesized by Integrated DNA Technologies (Coralville, IA) and cloned into pIRES-Neo3 (Takara Bio, Mountain View, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pIRES-Neo3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped viruses expressing SARS-CoV-2 S protein were generated by synthesizing the S gene which was truncated by 26 amino acids at the C-terminus, fused with HA tag by Integrated DNA Technologies, and cloned into plasmid pAbVec1 (Addgene, Watertown, MA), which were designated as pAbVec-SARS2-S.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pAbVec1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation of CRFK cells stably expressing human or animal ACE2: CRFK cells, plated the previous day, were transfected with pIRES-Neo-human (or cat, dog, cattle, horse, camel, hamster, rabbit, mink, white-tailed deer) ACE2-FLAG.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pIRES-Neo-human</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation of SARS-CoV-2 S pseudotyped viruses: The 2nd generation lentiviral packaging plasmid, psPAX2 (Addgene), a reporter plasmid pUCGFP-Luc (Addgene), and parental or mutant pAbVec-SARS2-S were transfected into HEK293 cells to produce pseudotyped viruses.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pUCGFP-Luc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pAbVec-SARS2-S</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SA/KRISP-K005325/2020 stock was subsequently passaged on Calu3 cells and NGS results showed this stock to contain only 13% of the furin site mutation; this stock was used for the in vitro virus replication kinetic experiments.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NGS</div><div>suggested: (PM4NGS, RRID:SCR_019164)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structures were analyzed and images created using PyMOL [92].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical analysis was performed using GraphPad Prism Software version 6 (San Diego, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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Results from scite Reference Check: We found no unreliable references.

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.08.25.457692: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The bound antibody was detected by AP conjugated goat- anti- human IgG Fc specific antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti- human IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 RBD plasmid cloning and protein expression: SARS-CoV-2 wildtype RBD (319-541aa) was cloned to pCDNA3.1 vector with 6 histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Delta variant (L452R_T478K) and Lambda variant (L452Q_F490S) structures were prepared by Coot software 26.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All protein structural figures are prepared by PyMOL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.25.457693: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Mouse experiments: Female 129S2 (catalog 287) and K18-hACE2 C57BL/6 (catalog 034860) mice were purchased from the Charles River and The Jackson Laboratory, respectively.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: All cells routinely tested negative for mycoplasma using a PCR-based assay.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-71 (Liu et al., 2021c) anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma, 12-349) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS2-57</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SARS2-71</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-S</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Millipore Cat# 12-349, RRID:AB_390192)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following blocking with FcγR antibody (BioLegend, clone 93), cells were stained on ice with CD45 BUV395 (BD BioSciences clone 30-F11), CD4 PE (BD BioSciences clone GK1.5)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD45</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero-TMPRSS2 cells were supplemented with 5 μg/mL of blasticidin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero-hACE2-TMPRSS2 cells were supplemented with 10 µg/mL of puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-hACE2-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Heterozygous K18-hACE2 C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) and 129 mice (strain: 129S2/SvPasCrl) were obtained from The Jackson Laboratory and Charles River Laboratories, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6J</div><div>suggested: RRID:MGI:3589388)</div></div><div style="margin-bottom:8px"><div>129S2/SvPasCrl</div><div>suggested: RRID:IMSR_CRL:287)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse experiments: Female 129S2 (catalog 287) and K18-hACE2 C57BL/6 (catalog 034860) mice were purchased from the Charles River and The Jackson Laboratory, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Homogenates then were analyzed for cytokines and chemokines by Eve Technologies Corporation (Calgary, AB, Canada) using their Mouse Cytokine Array/Chemokine Array 31-Plex (MD31) platform.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AB</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, mammalian cell codon-optimized nucleotide sequences coding for the soluble ectodomain of the S protein of SARS-CoV-2 including a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and hexahistidine tag were cloned into mammalian expression vector pCAGGS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_18926)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Graphs were generated using Graphpad Prism v9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, a TaqMan assay was designed to target a highly conserved region of the N gene (Forward primer: ATGCTGCAATCGTGCTACAA; Reverse primer: GACTGCCGCCTCTGCTC; Probe: /56-FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GACTGCCGCCTCTGCTC</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Probe</div><div>suggested: (UniPROBE, RRID:SCR_005803)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed on a BD LSRFortessa X-20 cytometer, using FlowJo X 10.0 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">QUANTIFICATION AND STATISTICAL ANALYSIS: Statistical significance was assigned when P values were < 0.05 using Prism Version 10 (GraphPad)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Limitations of study: We note several limitations in our study. (1) The studies in 129S2 mice precluded challenge with B.1.617.2, as it does not infect murine cells because it lacks an N501Y mutation. The generation of recombinant SARS-CoV-2 strains with spike genes encoding B.1.617.2 and an N501Y mutation could overcome this limitation. (2) Female 129S2 and K18-hACE2 mice were used to allow for group caging of the large cohorts required for these multi-arm vaccination studies. Follow-up experiments in male mice are needed to confirm results are not sex-biased. (3) We used lower vaccine dosing as a model for waning immunity. Studies that directly address durability of immune responses and protection are needed for corroboration. (4) We used historical, variant, or mixed mRNA vaccine formulations with homologous boosting schemes. Animals studies that test heterologous boosting (mRNA-1273 prime followed by mRNA-1273.351 boost) (Wu et al., 2021) also are needed to support clinical trials. (5) Our studies focused on immunogenicity and protection in two strains of mice because of the ability to set up large animal cohorts and the tools available for analysis. These results require confirmation in other animal models of SARS-CoV-2 infection including hamsters and non-human primates (Muñoz-Fontela et al., 2020). (6) We did not establish direct immunological correlates of vaccine protection or failure for all vaccine and challenge strain pairs. While some relationships were more pred...

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04927065</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study to Evaluate the Immunogenicity and Safety of mRNA-12…</td></tr></table>

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.08.25.21262569: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Serious adverse events (SAEs) and AEs of special interest were collected throughout each study.<br>IRB: All relevant ethical guidelines have been followed, all necessary IRB and/or ethics committee approvals have been obtained, all necessary patient/participant consent has been obtained and the appropriate institutional forms archived.<br>Consent: All relevant ethical guidelines have been followed, all necessary IRB and/or ethics committee approvals have been obtained, all necessary patient/participant consent has been obtained and the appropriate institutional forms archived.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 antigen was a stabilized pre-fusion spike protein ([2P], Δfurin, T4 foldon, His-tag), derived from the first clinical isolate of the Wuhan strain (Wuhan, 2019, whole genome sequence NC_045512), produced in ES-293 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ES-293</div><div>suggested: None</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04436276</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study of Ad26.COV2.S in Adults (COVID-19)</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04535453</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study to Evaluate a Range of Dose Levels and Vaccination I…</td></tr></table>

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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Results from scite Reference Check: We found no unreliable references.

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.08.24.457448: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Data collection and refinement: Diffraction data were collected at 100 K and at a wavelength of 0.9796 Å on the BL18U1 beam line of the Shanghai Synchrotron Research Facility.<br>IRB: This study was approved by the Institution Review Board of Tsinghua University (20210040)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Eight were male, and 1 was female.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: Cells were tested routinely and found to be free of mycoplasma contamination.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The blots were exposed to primary antibodies anti-β-Tubulin (CW0098, CWBIO), or anti-FLAG (F7425, Sigma) in 5% nonfat milk in 1× PBS containing 0.1% Tween 20 for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-β-Tubulin ( CW0098</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-FLAG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture: HEK293T (American Tissue Culture Collection, ATCC, Manassas, VA, CRL-3216), A549 (ATCC</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">#CCL-185), Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HeLa (ATCC #CCL-2) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, NY, USA) supplemented with 10% (vol/vol)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of SARS-CoV-2 pseudotyped virus: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G, Addgene #12259) using VigoFect (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2-lg, a recombinant Fc fusion protein of soluble human ACE2 (residues Gln18-Ser740), was expressed in 293F cells and purified using protein A affinity chromatography as described in our previous study36.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293F</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus-serum mixtures were incubated at 37°C for 30min, then added to A549-hACE2 cells in 96-well plates and incubated at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-hACE2</div><div>suggested: RRID:CVCL_A5KB)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids: The cDNAs encoding human or mink ACE2 were synthesized by GenScript and cloned into pLVX-IRES-zsGreen1 vectors (Catalog No. 632187, Clontech Laboratories, Inc) with a C-terminal FLAG tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-IRES-zsGreen1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of SARS-CoV-2 pseudotyped virus: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G, Addgene #12259) using VigoFect (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTG-MLV-Fluc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pTG-MLV-Gag-pol</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div><div style="margin-bottom:8px"><div>pMD2 . G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein crystallization: The gene encoding 333-527aa of SARS-CoV-2 RBD (WT or Y453F mutant) was cloned into the pFastbac-dual vector using the BamH1 and Hind3 restriction enzyme.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastbac-dual</div><div>suggested: RRID:Addgene_135584)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The two structures were determined using the molecular replacement method with Molrep in the CCP4 suite37, using the model of the human ACE2 and SARS-CoV-2 complex21.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CCP4</div><div>suggested: (CCP4, RRID:SCR_007255)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Subsequent model building and refinement were performed using COOT and PHENIX, respectively38,39.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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Results from scite Reference Check: We found no unreliable references.

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.08.22.457114: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-spike monoclonal antibodies from COVID-19 patients: The variable regions of anti-SARS-CoV-2 spike antibodies from COVID-19 patients were synthesized according to the published sequence (IDT) (Brouwer et al., 2020; Chi et al., 2020; Li et al., 2021; Robbiani et al., 2020; Suryadevara et al., 2021; Zost et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-spike</div><div>suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">donkey anti-mouse IgG Fc fragment antibody and APC-conjugated anti-human IgG Fc fragment specific antibody (Jackson ImmunoResearch, USA) were used.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometric analysis of antibodies: Plasmids expressing the full-length SARS-CoV-2 spike protein, Flag-NTD-PILR-TM and Flag-RBD-PILR-TM were co-transfected with the GFP vector into HEK293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 spike protein , Flag-NTD-PILR-TM</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2-stably transfected HEK293 cells (HEK293T-ACE2-transfectants) were reported previously (Liu et al., 2021b).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pcDNA3.4 expression vector containing the sequence that encodes the His-tagged extracellular domain of the spike protein was transfected into Expi293 cells and the His-tagged spike protein produced in the culture supernatants was then purified with a Talon resin (Clontech).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">48 hours later, B16F10 cells were washed twice with PBS,and then the cells were collected and frozen and thawed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>B16F10</div><div>suggested: NCI-DTP Cat# B16F10, RRID:CVCL_0159)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometric analysis of antibodies: Plasmids expressing the full-length SARS-CoV-2 spike protein, Flag-NTD-PILR-TM and Flag-RBD-PILR-TM were co-transfected with the GFP vector into HEK293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Balb/c female mice (7-weeks-old females) were purchased from SLC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Balb/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For Cryo-EM analysis, the sequence encoding the spike protein’s extracellular domain with a foldon and His-tag at the C-terminus (Cai et al., 2020) was cloned into a pcDNA3.4 expression vector containing the SLAM signal sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4</div><div>suggested: RRID:Addgene_131198)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transfection: A pME18S expression plasmid containing the full-length or subunit spike protein was transiently transfected into HEK293T cells using PEI max (Polysciences); the pMx-GFP expression plasmid was used as the marker of transfected cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pME18S</div><div>suggested: RRID:Addgene_52384)</div></div><div style="margin-bottom:8px"><div>pMx-GFP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cDNA of the variable regions of the heavy chain and light chain were cloned into a pCAGGS vector containing sequences that encode the human IgG1 or kappa constant region.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_18926)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The primers for mutagenesis were designed on Agilent’s website (https://www.agilent.com/store/primerDesignProgram.jsp).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Agilent’s</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transfection: A pME18S expression plasmid containing the full-length or subunit spike protein was transiently transfected into HEK293T cells using PEI max (Polysciences); the pMx-GFP expression plasmid was used as the marker of transfected cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Polysciences)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies binding to the GFP-positive cells were shown in the figures using FlowJo software (BD bioscience).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PRNT50 neutralization titers for vaccinated sera were determined using 3-parameter nonlinear regression curve (GraphPad Prism).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For automated data acquisition, SerialEM software was used to collect cryo-EM image data.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction: All of image processes were carried out on cryoSPARC software (Punjani et al., 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The corrected model was refined by the phenix.real_space_refine program (Liebschner et al., 2019) with secondary structure and Ramachandran restraints, then the resulting model was manually checked by COOT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This iterative process was performed for several rounds to correct remaining errors until the model was in good agreement with geometry, as reflected by the MolProbity score of 2.07 (Williams et al., 2018).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MolProbity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data and statistical analysis: FlowJo version 10.7 (BD Biosciences, USA) was used to analyze the flow cytometry data, and Graphpad Prism version 7.0e was used for graph generation and statistical analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

I highlighted the entire title and hopefully this link goes to this specific video (sometimes it just goes to YouTube). I then right clicked at a specific time and pasted the URL into this textbox, and that allows me to share the video at a time I am interested in. https://youtu.be/OB0Ki50WkWk?t=370 I then used the tag of my LibreText page (which must be public) to aggregate it (f21c1403c11) on the chapter summary page. (This feature is only available on the first page of each chapter, which has links to all the topics of the chapter)

There's something here that sounds like the idea of a friendship book, but in online/blog form.

It's also a bit reminiscent of a social startup in the late 00s called Formspring.me.

Everything old is new again?

new tag?: makes zero practical sense

makes zero practical sense because there exists no runtime representation of the type

Links and References:

• Who's in Charge? Free Will and the Science of the Brain
• Avoiding Falling Victim to The Narrative Fallacy
• The Revised Psychology of Human Misjudgment, by Charlie Munger
• Influence: The Psychology of Persuasion
• The Munger Two Step

Soumen's work in virtual world design is elegant; complex interactions connecting users and components with (and thru) data..

Question: Are these tools ready for prime time?

Note: First use of'resources' tag?

See you soon, Later

Check that not in cloud 1st. Can't I just delete from cloud as well?

SciScore for 10.1101/2021.08.17.456704: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Study approval: Mouse care and experimental procedures with mice were performed under pathogen-free conditions in accordance with established institutional guidance and approved Animal Care and Use Protocols (ACUP) from the Research Animal Care Committee at Sorrento Therapeutics Inc.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Mice and cell culture: For intramuscular antigen challenge, C57BL/6 female mice 5-6 weeks old were injected with either 100 μg RBD-C-tag (300 μg adjuvant) or 50 μg RBD-C-tag (150 μg adjuvant) intramuscularly.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Detection of RBD and Spike binding serum IgG antibodies by ELISA: A direct binding ELISA format was used to detect the anti-SARS-COV-2 RBD or Spike IgG antibody in mouse serum samples.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Spike binding serum IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-COV-2 RBD or Spike IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">donkey anti-mouse IgG conjugated to AlexaFluor 647 (Invitrogen #A-31571) or IL-10 APC antibodies ( eBioscience, # 17-7101-81).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>donkey anti-mouse IgG</div><div>suggested: (Molecular Probes Cat# A-31571, RRID:AB_162542)</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Molecular Probes Cat# A-31571, RRID:AB_162542)</div></div><div style="margin-bottom:8px"><div>IL-10 APC</div><div>suggested: (Thermo Fisher Scientific Cat# 17-7101-81, RRID:AB_469501)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Human parental HEK293 cells (ATCC, CRL-1573) were cultured in DMEM supplemented with 10% FBS and antibiotics/antimycotics (Gibco).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Simian VeroE6 cells were plated at 18×103 cells/well in a flat bottom 96-well plate in a volume of 200 μl/well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Longitudinal near infrared in vivo imaging of lymphatics: IRDye800-RBD-C-tag was injected intramuscularly into the biceps femoris of C57BL/6 mice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.18.456855: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Authentication: To monitor growth kinetics, the cells were seeded on 96-well plates, treated as indicated and subjected to CellTiter-Glo luminescent cell viability assay (Promega) according to the manufacturer’s instructions.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies used: anti-puromycin antibodies (Millipore Cat#MABE343),</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-puromycin</div><div>suggested: (Millipore Cat# MABE343, RRID:AB_2566826)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mRNA library preparation for MARS-seq: Total RNA was isolated from HEK293T cells using Bio Tri RNA reagent (Bio-lab) and mRNA was captured using Oligo d(T)25 magnetic Beads (NEB) according to manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Biological resources: Cultured cell lines: MCF7 and HEK293 cells were from ATCC, MRC5 and Vero E6 cells were generously provided by Zvi Livneh and Yosef Shaul, respectively (WIS, Israel).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MCF7</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus stocks were propagated (four passages) and titered on Vero E6 cells (Vero E6, ATCC® CRL-1586™).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reagents: DNA constructs: To create HA-tagged NSP1 lacking viral 5’UTR for expression in cell culture (pCRUZ-HA-NSP1), NSP1 was obtained from the Forchheimer plasmid bank (WIS, Israel) and cloned into pCRUZ-HA (sc-5045) plasmid (Santa Cruz Biotechnology) using restriction-free cloning and primers #1,2 (here and on, see Table S1 for primers’ sequences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCRUZ-HA-NSP1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCRUZ-HA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To create HA-tagged NSP1 with the viral 5’UTR for expression in cell culture (pcDNA3.1-TSS-5’UTR-HA-NSP1), HA-NSP1 was PCR-amplified from the pCRUZ-HA-NSP1 plasmid using primers #5,6 and introduced immediately after the transcription start site of the pcDNA3.1(-) plasmid using restriction-free cloning.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-TSS-5’UTR-HA-NSP1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">reporter gene bearing Hisx6 tag was amplified using primers #18,19 and cloned between HindIII-KpnI sites of pcDNA5/FRT/TO plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5/FRT/TO</div><div>suggested: RRID:Addgene_19445)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting product was used in a PCR to amplify pcDNA3.1(-) plasmid as template.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1 ( - )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nsp1 protein purification for in vitro assay: The pET28 plasmid encoding for His-tagged NSP1 was transformed by heat shock into BL-21 E.coli and a single grown colony was inoculated into 5ml of LB supplemented with Kanamycin (50μg/ml) and incubated shaking at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28</div><div>suggested: RRID:Addgene_21766)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mRNA library preparation for MARS-seq: Total RNA was isolated from HEK293T cells using Bio Tri RNA reagent (Bio-lab) and mRNA was captured using Oligo d(T)25 magnetic Beads (NEB) according to manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Oligo</div><div>suggested: (oligo, RRID:SCR_015729)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After treatment with ECL reagent (Azure Biosystems), images were captured using Licor Fc imaging system and the signal intensities were calculated using ImageStudio software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageStudio</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Corrected counts were normalized by mouse PolyA+ enriched RNA, which was mapped to mouse genome using STAR (17) not allowing mismatches.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.18.456769: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibodies used were anti-GFP HRP-DirecT (#598-7, MBL, Nagano, Japan), anti-polyhistidine-tag (#PM032, MBL), and anti-mCherry (#Z2496N, TaKaRa, Shiga, Japan)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-GFP</div><div>suggested: (MBL International Cat# 598-7, RRID:AB_10597267)</div></div><div style="margin-bottom:8px"><div>#598-7 , MBL , Nagano , Japan) , anti-polyhistidine-tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mCherry</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For observation of lysosome localization, mCherry-tagged protein-expressing Neuro2a cells were incubated in a medium containing 0.5 μM Lysotrascker green DND-26 (Thermo Fisher) for 30 min before image acquisition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Neuro2a</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To construct an expression plasmid, cytoplasmic region- and transmembrane-lacking hACE2 tagged with a monomeric variant of eGFP carrying the A206K mutation (hACE2-eGFP), the fragment encoding hACE2, was inserted into pmeGFP-N1 with NheI and EcoRI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hACE2</div><div>suggested: RRID:Addgene_1786)</div></div><div style="margin-bottom:8px"><div>pmeGFP-N1</div><div>suggested: RRID:Addgene_27766)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A synthetic oligonucleotide for the ER-targeting signal peptide was inserted into pmCherry-N1 (pER-mCherry) with NheI and AgeI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmCherry-N1</div><div>suggested: RRID:Addgene_87327)</div></div><div style="margin-bottom:8px"><div>pER-mCherry</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fragments were inserted into pER-mCherry-N1 with XhoI and BamHI (pER-mCherry-S1 and pER-mCherry-S1-2, respectively).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pER-mCherry-N1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pER-mCherry-S1-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The whole sequences of the coding regions of pER-mCherry-S1 and pER-mCherry-S1-2 are presented in Supplemental Figure.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pER-mCherry-S1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids encoding hACE2-eGFP, ER-mCherry-S1, or ER-mCherry-S1-2 (16 μg) and sonicated salmon sperm DNA (14 μg) were transfected into the cells with 240 μL of polyethyleneimine (#43896,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hACE2-eGFP</div><div>suggested: None</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.08.17.456689: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Western blot: Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously (50).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-COV-2 S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma-Aldrich, St. Louis, MO) was used as a secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure binding of a full-length S protein to monoclonal antibodies, the antibody was immobilized to anti-human IgG Fc Capture (AHC) biosensor (ForteBio, Fremont, CA) following a protocol recommended by the manufacturer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Control sensors with no S protein or antibody were also dipped in the ACE2 or S protein solutions and the running buffer as references.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For ACE2615-foldon T27W staining, APC conjugated anti-HIS antibody (Miltenyi Biotec, Auburn, CA) was used as secondary antibody at 1:50 dilution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HIS</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, Expi293F cells transfected with monomeric ACE2 or dimeric ACE2 expression construct and the supernatant of the cell culture was collected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Murine Leukemia Virus (MLV) particles (plasmids of the MLV components kindly provided by Dr. Gary Whittaker at Cornell University and Drs. Catherine Chen and Wei Zheng at National Center for Advancing Translational Sciences, National Institutes of Health), pseudotyped with various SARS-CoV-2 S protein constructs, were generated in HEK 293T cells, following a protocol described previously for SARS-CoV (51, 52).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To prepare for infection, 7.5×103 of HEK 293 cells, stably transfected with a full-length human ACE2 expression construct, in 15 μl culture medium were plated into a 384-well white-clear plate coated with poly-D-Lysine to enhance the cell attachment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T/17</div><div>suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293T cell line stably overexpressing the human ACE2 cell surface receptor protein was kindly provided by Drs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The S genes were fused with a C-terminal twin Strep tag (SGGGSAWSHPQFEKGGGSGGGSGGSSAWSHPQFEK) and cloned into a mammalian cell expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-IRES-puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV DR8.2, and luciferase reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pHR’</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression constructs: Genes of full-length spike (S) protein from Gamma (hCoV-19/Brazil/AM-992/2020; GISAID accession ID: EPI_ISL_833172), Kappa (hCoV-19/India/MH-NEERI-NGP-40449/2021; GISAID accession ID: EPI_ISL_1547802) and Delta (hCoV-19/India/GJ-GBRC619/2021; GISAID accession ID: EPI_ISL_2020954) were synthesized by Twist Bioscience (South San Francisco, CA) or GENEWIZ (South Plainfield, NJ).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GENEWIZ</div><div>suggested: (GENEWIZ, RRID:SCR_003177)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Kd was obtained by fitting Req value and its corresponding concentration to the model: “one site-specific” using GraphPad Prism 8.0.2 according to H.J. Motulsky, Prism 5 Statistics Guide, 2007, GraphPad Software Inc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated data collection was carried out using SerialEM version 3.8.6 (53) at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.825 Å) at an exposure rate of 20.24 (for Delta), ∼20.69/20.63/27.13 (for three data sets of Gamma), or ∼21.12/20.10 (for two data sets of Kappa) electrons per pixel per second.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstructions: Drift correction for cryo-EM images was performed using MotionCor2 (54), and contrast transfer function (CTF) was estimated by Gctf (55) using motion-corrected sums without dose-weighting.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MotionCor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Density maps were corrected from the modulation transfer function of the K3 detector and sharpened by applying a temperature factor that was estimated using post-processing in RELION.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Several rounds of manual building were performed in Coot (57).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

A caveat is that all our experiments were performed in vitro; additional studies with authentic viruses will be needed to confirm our findings in more clinically relevant settings. If our hypothesis is valid, what is the structural basis for the enhanced fusogenicity of the Delta S protein? All mutations but one in the Delta S are located in either the RBD or NTD. Our extensive binding studies indicate that the Delta S does not engage the receptor ACE2 more tightly than does any other variant. It is unclear what other functional roles the NTD may play in the membrane fusion process, besides protecting the nearby RBDs. If the mutations in the NTD enhance RBD exposure to potential receptors, we should have observed, in our cryo-EM study, more particles in the RBD-up conformation from the Delta data set. Thus, the structural changes in both the RBD and NTD are unlikely to explain the efficient membrane fusion by the Delta variant. The last mutation, D950N, is unique to Delta and located in HR1 of S2 near the FPPR. D950N eliminates a negative charge (three in a trimer), but we have not observed any obvious structural changes caused by this substitution in the prefusion conformation. Its location nonetheless appears to be a critical site that can influence the refolding of S2, required for membrane fusion. Although D950 is not involved in a salt bridge in the G614 trimer, it is conceivable that the local change in the electrostatic potential may destabilize the prefusion S2 in a v...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 35. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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§ 4 (2) Krankenhaus-Covid-19-Verordnung

§ 3 (1) Krankenhaus-Covid-19-Verordnung

§ 3 (1) Krankenhaus-Covid-19-Verordnung

SciScore for 10.1101/2021.08.13.454991: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: In vivo IAV infection model: All mice were housed under specific-pathogen-free conditions at Seattle Children’s Research Institute and all animal experiments performed at Seattle Children’s Research Institute were approved by the Institutional Animal Care and Use Committee (IACUC00580).<br>IRB: The project was submitted to the French Ethics Committee CEEA (Comité d’Ethique en Expérimentation Animale) and obtained the authorization APAFIS#10108-2017060209348158 v3.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">A pilot experiment showed a protective effect of C910 in male animals therefore we used male mice for further experiments (data not shown).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Cells in each condition were randomly selected and analyzed under the same threshold.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies and reagents: C910 was purchased from Chembridge (ID: 5454910, San Diego, CA, USA) or Synthenova (ID: SN0218L3, Hérouville Saint-Clair, France).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C910</div><div>suggested: (GenWay Biotech Inc. Cat# GWB-82C910, RRID:AB_10513344)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, mouse anti-Rac1 (610651 [clone 102]), mouse anti-Rab4 (610888), mouse anti-Lamp1 (611043) was from BD Bioscience; mouse anti-TGN38 (sc-101273), rabbit anti-MEK2 (SC-524) from Santa Cruz; rabbit anti-Calnexin (ADI-SPA-860-D) from Enzo; mouse-anti-LBPA (Z-PLBPA) was from Echelon Biosciences; Paraformaldehyde (PFA) (15710) from Electron Microscopy science.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Calnexin</div><div>suggested: (Enzo Life Sciences Cat# ADI-SPA-860-D, RRID:AB_2038898)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Horseradish peroxidase (HRP)-conjugated goat anti-mouse (P0399) or swine anti-rabbit secondary antibodies (P0447) were from Dako.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse ( P0399</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were next labelled with an anti-Rac1 mouse antibody [clone 102] for 1 h and revealed with Alexa Fluor 488-coupled anti-mouse secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In parallel, cell lysates from intoxicated cells were examined for glucosylated-Rac1 that no longer react to anti-Rac1 monoclonal antibody [clone 102].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Rac1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Signals were revealed using horseradish peroxidase-conjugated goat anti-mouse or swine anti-rabbit secondary antibodies (DAKO) followed by chemiluminescence using Immobilon® Western (Millipore).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HeLa, L929, Vero, A549, Vero E6 and U2OS-ACE2 cell lines were cultured at 37°C in 5% CO2 in DMEM/GlutaMax (Invitrogen) supplemented with 10% heat-inactivated FBS (F9665, Sigma-Aldrich) and 1% penicillin/streptomycin (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>U2OS-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cytotoxic effects of the LCGTs TcdA and TcdB on Vero cells (Sigma-Aldrich) were evaluated by measure of cell rounding.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IAV infection: For single-cycle IAV infection assays, A549 cells (70,000/ well) were infected with a reporter H1N1 A/WSN/33 (H1N1WSN PB2-2A-mCitrine), at a MOI of 5 PFU/cell for 6 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For multicycle growth assays, A549 cells were infected with a seasonal A/Bretagne/7608/2009 (H1N1pdm09) strain adapted to human cell lines (50), at the MOI of 10−3 infective particles/cell and the production of infectious particles in the culture supernatant was determined at 24 h using a standard plaque assay on the highly sensitive canine MDCK-SIAT cells, as described previously(72).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MDCK-SIAT</div><div>suggested: ECACC Cat# 05071502, RRID:CVCL_Z936)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Determination of CC50s of C910 on HUVECs and HeLa cells: Cells seeded in Corning™ 96 well Flat Clear Bottom Black Microplates (#3603) were incubated with increasing doses of C910 for 6 h, medium was replaced with fresh medium including 10% of Alamar Blue (final concentration 1:10).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Medisch Centrum Utrecht, Netherland), HUVECs were fixed at room temperature for 2 h in 2.5% glutaraldehyde in PHEM buffer, pH 7.2 (60 mM 1,4 piperazine diethylsulfonic acid (PIPES), 25 mM N-2-hydroxyethylpiperazine</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HUVECs</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral challenges were carried out on groups of 9~10 week-old C57BL/6 male mice (Charles River Laboratories).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Experiments were conducted on adult male C57BL/6J mice (23.5 ± 0.3 g) purchased from Janvier Labs (Le Genest St Isle, France).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6J</div><div>suggested: RRID:IMSR_JAX:000664)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and bacterial toxins: Human umbilical vein endothelial cells (HUVECs) (PromoCell, Heidelberg, Germany) were cultured as described (24).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PromoCell</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HIS-tagged B subunit of Shiga toxin 1(SML0655) and Apilimod (A149227) from Sigma; rabbit anti-EEA1 (#3288), rabbit anti-Rab7 (#9367), rabbit-anti-his tag (#12698) from Cell Signaling; rabbit anti-Rab7 (ab137029), mouse anti-Giantin (ab37266) and Cathepsin B Activity Assay Kit (ab65300) from Abcam; mouse-anti-6x-his (R930-25 [clone 3D5]), DQTM Red BSA (D-112051) from ThermoFisher Scientific; mouse-anti-EEA1 (BD610457), mouse-anti-Rab5 (610724)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher Scientific</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, mouse anti-Rac1 (610651 [clone 102]), mouse anti-Rab4 (610888), mouse anti-Lamp1 (611043) was from BD Bioscience; mouse anti-TGN38 (sc-101273), rabbit anti-MEK2 (SC-524) from Santa Cruz; rabbit anti-Calnexin (ADI-SPA-860-D) from Enzo; mouse-anti-LBPA (Z-PLBPA) was from Echelon Biosciences; Paraformaldehyde (PFA) (15710) from Electron Microscopy science.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Bioscience</div><div>suggested: (BD Biosciences, RRID:SCR_013311)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">8 software (GraphPad, San Diego, CA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed with FlowJo (BD Bioscience).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fiji ImageJ software (National Institutes of Health) was used for image processing and quantification.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, each individual cell was selected from a single mid-z stack of the confocal image by hand-drawing a “region of interest” (ROI) from Fiji software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji</div><div>suggested: (Fiji, RRID:SCR_002285)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Measures of the size of EEA1-positive vesicles were performed from the single mid-z stack of confocal images that were next quantified with the Analyze particles tool from Fiji Software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Analyze</div><div>suggested: (ANALYZE, RRID:SCR_009120)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were fitted with Prism v8 software (Graphpad Inc., San Diego, CA) to obtain the concentrations giving 50% toxicity.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 57 and 50. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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Results from scite Reference Check: We found no unreliable references.

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<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.11.455956: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: This study was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 2 h, infected cells were washed an additional five times with DMEM prior to adding media supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) to reduce parental background.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">An anti-S2 SARS-CoV-2 S polyclonal primary antibody (1:1,500 dilution,Invitrogen PA5-114534) and an Alexa Fluor 680-conjugated goat anti-rabbit secondary antibody (1:20,000 dilution, Jackson Laboratory 111-625-144) were used for Western-blotting.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-S2 SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">At this stage, excess DMEM was removed from the cells and 40 µL from each well (containing sera and pseudovirus) was transferred to the 96-well plate seeded with HEK-293T cells expressing hACE2 and incubated at 37°C for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 S ectodomain with VFLIP mutations (48), the native furin cleavage site (RRAR), and B.1.617.2 spike mutations (T19R, G142D, E156G, T478K, and D950N substitutions and a deletion of residues 157 and 158) was synthesised by GenScript into pCMV with a C-terminal avi tag followed by an octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_20783)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 S ectodomain with hexapro mutations and P.1 spike mutations (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F) was synthesised by GenScript into pCMVR with foldon, a C-terminal avi tag followed by an octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVR</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">2) were obtained from GISAID (using outbreak.info) and plotted using GraphPad PRISM software (version 9.2.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad PRISM</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative luciferase units were plotted and normalized in Prism (GraphPad): cells alone without pseudovirus was defined as 0 % infection, and cells with virus only (no sera) was defined as 100 % infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Prism (GraphPad) nonlinear regression with “[inhibitor] versus normalized response with a variable slope” was used to determine IC50 values from curve fits.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Particles in well-formed 3D classes were then used for local refinement in cryoSPARC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The model was then refined and rebuilt into the map using Coot (89), Rosetta (90, 91), Phenix (92), and ISOLDE (93).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model validation and analysis used MolProbity (94), EMringer (95), Phenix (92) and Privateer (96).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MolProbity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.08.455468: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To test blocking, recombinant human ACE2 (Gln18-Ser740, C-terminal His-tag) from RayBiotech (∼3.8 µM in measurement buffer, # 230-30165-100 distributed by antibodies-online GmbH, # ABIN6952473) was flushed into the flowcell and shortly incubated before the measurement.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>His-tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>antibodies-online GmbH,</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Experimental Procedures, and Data Analysis: SARS-CoV-1 and SARS-CoV-2</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (BioLegend Cat# 946101, RRID:AB_2892515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Equilibrium measurements with MT and rupture experiments with an AFM were evaluated with custom MATLAB and Python scripts to deduce force stability and kinetics.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div><div style="margin-bottom:8px"><div>Python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.07.455523: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal experiments were approved by the Animal Care and Use Committee of Wuhan University.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Age-matched (9-10-week-old) female mice were grouped for infection of nanobodies (0.5mg/kg).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">After 2–3 rounds of selection-amplification cycle, single colonies were randomly selected into deep 96-well culture plate containing 850 μL/well of 2YT (100 μg/mL ampicillin) and shook at 37°C for 3h.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD-His binding to the plate was detected with anti-His tag mouse monoclonal antibody (1:3000 dilution, SinoBiological, 105327-MM02T) and followed by an HRP conjugated anti-mouse IgG (H+L) Goat antibody (Beyotime, A0216).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His tag</div><div>suggested: (Sino Biological Cat# 105327-MM02T, RRID:AB_2857924)</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Beyotime Cat# A0216, RRID:AB_2860575)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For IFA, Anti-hACE2 antibody and anti-SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (Cat: 10108-RP01 and 40143-MM05, SinoBiological) were added as primary antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-hACE2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV/SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines and viruses: Vero-E6 (ATCC® CRL-1586), CaCO2(ATCC® HTB-37) and 293T (ATCC® CRL-3216) cell are cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermal Fisher, # 12430112), supplied with 10% fetal bovine serum (Thermal Fisher, # 26140079),1%</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 (ATCC number: CRL-1586) cells were cultured to determinate viral titer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protection of K18-hACE2 transgenic mice against SARS-CoV-2: K18-hACE2 transgenic mice expressing human ACE2 driven by the human epithelial cell cytokeratin-18 (K18) promoter, were purchased from Gempharmatech and housed in ABSL-3 pathogen-free facilities under 12-h light-dark cycles with ad libitum access to food and water.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The whole coding cassette was ligated into a pCMV3 expression vector with a signal peptide of MEFGLSWVFLVALFRGVQC at the N-terminal, and either a 6-his tag (for RBD-his) or a human IgG1 Fc fragment with (GSSSS)3 linker at C-terminal.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3</div><div>suggested: RRID:Addgene_161029)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, pseudovirus bearing wildtype SARS-CoV-2 S protein or mutants were produced by co-transfection with plasmids expressing corresponding protein and backbone plasmid pNL-4-3-Luc.-</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNL-4-3-Luc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To accurately quantify the absolute number of SARS-CoV-2 genomes, a standard curve was prepared by measuring the SARS-CoV-2 N gene constructed in the pCMV-N plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-N</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ExpiCHO Expression System was purchased from Thermal Fisher (#A29133).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Thermal Fisher</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein expression and purification: The constructs of VHHs were selected from phage display, RBD fragment (aa319-541) of SARS-CoV-2 S protein (GenBank: MN908947.3) was synthesized by Genewiz Inc (GENEWIZ, Suzhou, China), the extracellular domain of human ACE2 (1-740 aa) (GenBank: NM_021804.1) was amplified from a plasmid (HG10108-ACG, Sinobiologic, Beijing, China).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GENEWIZ</div><div>suggested: (GENEWIZ, RRID:SCR_003177)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The acid eluted fraction was neutralized with 1M Tris-HCl, pH9.0 and was concentrated and desalted into PBS with Amicon® Ultra-15, PLTK Ultracel-PL membrane (MilliporeSigma Life Science Center, Burlington, Massachusetts, USA) with appropriate MWCO.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Amicon®</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The IC50 values were calculated with non-linear regression using GraphPad Prism 8 (GraphPad Software, Inc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protection of K18-hACE2 transgenic mice against SARS-CoV-2: K18-hACE2 transgenic mice expressing human ACE2 driven by the human epithelial cell cytokeratin-18 (K18) promoter, were purchased from Gempharmatech and housed in ABSL-3 pathogen-free facilities under 12-h light-dark cycles with ad libitum access to food and water.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gempharmatech</div><div>suggested: (GemPharmatech, RRID:SCR_017239)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All data was analyzed by XLfit (IDBS, Boston, MA 02210) or Prism 5(GraphPad Software, San Diego, CA 92108).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.10.455627: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All protocols were approved by the Institutional Animal Care and Use Committees of Guangzhou Customs District Technology Center and Guangzhou Medical University.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bound antibodies were detected with Peroxidase conjugated goat anti-human kappa light chains antibody (A7164-1ML, Sigma).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human kappa light chains</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Affinity measurement of antibodies by Surface plasmon resonance (SPR): SPR experiments were all conducted with a Biacore T200 system (GE Healthcare); All assays were performed with a Sensor Chip Protein A (GE healthcare),with a HBS EP+ running buffer (0.1M HEPES, 1.5M NaCl, 0.03M EDTA supplemented with 0.005% vol/vol surfactant P20 at 25°C.) To determine the affinities of nanobody VHH18, human IgG1 antibody 2F8, and 2022 to SARS-CoV-2 RBD-His tag, spike trimer-His tag and other S1-His tag variants, antibodies were immobilized onto the sample flow paths of the sensor Protein A chip.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sensor was saturated with the first antibody, either 2F8 Fab or VHH18-His, subsequently, the above bioprobes were flown over with the different second antibody, either VHH-his or 2F8 Fab, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VHH18-His</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were tested with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, SinoBiological, Inc.), and an HRP-labelled goat anti-rabbit as secondary antibody (111-035-003, Jackson ImmunoResearch).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 nucleocapsid protein</div><div>suggested: (Proteintech Cat# 67666-1-Ig, RRID:AB_2882862)</div></div><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID:AB_2313567)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293 (ACS-4500TM, ATCC) and African Green monkey kidney-derived VeroE6 cells (CRL-1587, ATCC) were cultured and passaged in DMEM with 10% FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACS-4500TM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transiently transfected to HEK293 cells to obtain recombinant RBD alanine mutants and purified by protein A columns.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293-hACE2 was constructed and sorted by Bio-Thera Solutions, Ltd. Recombinant Proteins: Biotinylated 2019-nCoV S protein RBD, His,Avitag™ (SPD-C82E9,Acrobiosystems); SARS-Vov-2 S protein RBD, His Tag (SPD-S52H6,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>S protein RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Balb/c mice were mildly anesthetized with isoflurane and i.n. transduced with 2.5×109 PFU of Ad5-ACE2 in 75ul DMEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Balb/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Values were determined using four-parameter logistic regression (GraphPad Prism).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

---

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

Results from JetFighter: We did not find any issues relating to colormaps.

---

Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

---

Results from scite Reference Check: We found no unreliable references.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.08.11.455921: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">interferon gamma (IFNγ) (SRP3058) and human Interleukin-4 (IL-4) (I4269) were from Sigma-Aldrich, UK; Primary antibodies were as follows: anti-CTGF goat polyclonal antibody (sc-14939), and anti-COL3A1 goat polyclonal antibody (sc-8781) were from Santa Cruz Biotechnology,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IL-4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CTGF</div><div>suggested: (Santa Cruz Biotechnology Cat# sc-14939, RRID:AB_638805)</div></div><div style="margin-bottom:8px"><div>anti-COL3A1</div><div>suggested: (Santa Cruz Biotechnology Cat# sc-8781, RRID:AB_638604)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibodies were as follows: polyclonal rabbit anti-goat Immunoglobulins/HRP (P0160) and polyclonal swine anti-rabbit Immunoglobulins/HRP (P0217) were from Dako, DK; Cy™3-conjugated</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-goat</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>P0160</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Agilent Cat# P0217, RRID:AB_2728719)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako, Denmark), diluted at 1:2500 in PBSTM were added for approx.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HRP)-conjugated secondary antibodies ( Dako , Denmark)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody concentration varied and depended on the antibody used, but it was usually at 1:200 or similar for the primary antibodies (i.e. TLR4) and 1:400 for ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>TLR4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies used were mouse anti-human TLR4 and rabbit anti-spike S1 with the appropriate secondary antibodies (anti-rabbit-minus and anti-mouse-plus PLA probes) from the kit and red detection reagents. 293-hTLR4-HA cells: 293-hTLR4-HA cells (InvivoGen, USA) are HEK 293 cells stably transfected with the hTLR4a gene fused at the 3’ end to an influenza haemagglutinin (HA) tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human TLR4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit-minus</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse-plus PLA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies used were mouse anti-human TLR4 and rabbit anti-spike S1 with the appropriate secondary antibodies (anti-rabbit-minus and anti-mouse-plus PLA probes) from the kit and red detection reagents. 293-hTLR4-HA cells: 293-hTLR4-HA cells (InvivoGen, USA) are HEK 293 cells stably transfected with the hTLR4a gene fused at the 3’ end to an influenza haemagglutinin (HA) tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293-hTLR4-HA</div><div>suggested: RRID:CVCL_Y394)</div></div><div style="margin-bottom:8px"><div>HEK 293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293 cells express very low levels of endogenous TLR4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">THP-1 cells and differentiation: The human monocytic cell line THP-1 was routinely maintained in RPMI 1640 (1x) growth medium containing 10% of heat-inactivated FBS, 4.5 mg/ml D-glucose, 2mM L-glutamine, 10mM HEPES, 1mM pyruvate, 0.05 mM 2-mercaptoethanol, and 1% Penicillin/streptomycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>THP-1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phorbol 12-myristate 13-acetate (PMA) (1201/1) was from Bio-Techne, UK; Cytokines:</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Cytokines</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis was performed and graphs were produced using GraphPad Prism 8, either using a Kruskal Wallis test, Wilcoxon Signed Rank Test, or Repeated Measures One-way ANOVA with G-G correction followed by Tukey’s multiple comparisons post hoc test as appropriate to the experiment and as indicated in the figure legends.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 25. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

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Results from scite Reference Check: We found no unreliable references.

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