[TEST] Reviewer 2: In their manuscript “Release from cell cycle arrest with Cdk4/6 inhibitors generates highly synchronised cell cycle progression in human cell culture” Trotter and Hagan investigated cell synchronization via CDK4 inhibitors as an alternative approach to commonly used thymidine-release regimes. The authors find that CDK4 inhibitor-based synchronization is suitable in 5 out of 25 tested cell lines (they show only data of 4 cell lines!) and provide FACS and Western blot data confirming this notion. Overall, the manuscript has the character of a methods study and does not explore the consequences and potential side-effects of CDK4 inhibitor based synchronization beyond the first two mitoses. Using CDK4 inhibitors to (pre)-synchronize cells in G1 phase is not a novel idea and has been used/published before (e.g. recently by Jackman et al., JCB 2020), however, to my knowledge a methodological study as presented here comparing different cell lines and different CDK4 inhibitors to synchronize cells has not been performed. Precise and as much as possible perturbation-free cell cycle synchronization remains key to cell cycle research, hence I think the presented manuscript has the potential to be a valuable addition to the field.

Except for the missing information on the number of experimental repeats for most experiments (see below) and a request to also include a tabular overview of all cell lines investigated, I do not have major criticisms precluding publication from my point of view. There are several points, however, the authors should address to make the manuscript stronger and more useful to the community.

1) Discussion & Introduction

-In recent years, live imaging studies of asynchronous cells, e.g. by the Meyer, Spencer, Mansfeld, Purvis or Bakal labs, have demonstrated that at least for single cell analyses no synchronization regimes are required anymore because singe cells tracks can be aligned computationally to a common cell cycle starting point after the experiment. Of course, such approaches are not compatible with biochemical experiments, but certainly support diverse functional analyses that previously were only possible by synchronized populations such as monitoring degradation/expression profiles of proteins. Hence, to my opinion at least the introduction would benefit from a short paragraph putting the “old school” bulk synchronization regimes to which also their approach belongs into context with such single cell alternatives.

-Can the authors provide a reference/results for their statement that RPE-1 cells are “refractory to double thymidine block induction synchronization” and that the levels of synchrony are low after serum starvation (l251ff)?

-Contact inhibition as an alternative method for synchronization, especially in RPE-1 cells, should be mentioned as well.

-p21, l516ff and p22, l534ff. Can the authors please indicate the names of the cell lines they refer to and not just the references. Also, does this argumentation then hold true for the altered genotypes we know of the non-responsive cell lines? E.g. MCF10A, which lack p16 and amplify myc signaling? Here, it would make sense to include known restriction-point genotype information in the table of all cell lines tested as suggested below. This would greatly benefit researchers choosing the right synchronization protocol for their individual cell line models.

-p23, l580ff. The closing statement is weak and does not connect well to the text before. Could be completely omitted.

-p20, l494. While the authors show based on gH2AX staining that there is not increased DNA damage due to CDK4i synchronization in S phase and mitosis they have no own data showing that chromosome segregation is not affected by CDK4 inhibition. While they reference a personal communication with the Pines lab, they should tone down this claim sufficiently.

P19, l474. The authors should remove the claim of novelty, because CDK4 inhibitors have been used before to synchronize cells, e.g. by refs 56 and 57 and others, e.g. Spencer et al., 2013 have use MEKK inhibitors, which target CDK4 indirectly via cyclin D.

P20, l501. This holds also true for contact inhibition!

2) Figures

-For data presentations with bar charts showing lower n (<10) it would be useful if the authors over plot the single data points on top of the bar charts to enable the reader estimating the distribution of the data. Also, the precise number of repetitions should be indicated, e.g. Fig 1, 4, 6,7,8, 9. It should be indicated if bars show the mean or median!

-All Western blot analyses should indicate molecular markers (Figs 3 &5) and in the absence of quantifications, it should be mentioned of how many experiments the data is representative of.

-Generally, for all experiments/data it should be indicated of how many independent repeats the data is representative/shown. E.g. it is not clear of how many repeats the data without error bars is representative of in basically all figures with such data.

-Generally, the authors should carefully re-read their figure captions and make sure that the referrals to explanations in other figures are correct and provide sufficient data. As a reader, I find it somewhat cumbersome, if I have read the legends of 3 figures to get all the required information – e.g. legend to figure 8 refers to figure legend 7, which refers to Figure legend …

-Figure 9, the unit of the concentrations of palbociclib is not given.

Experiments

-Western blot analyses in Figures 3 and 5. I am not sure if I understand the data and the corresponding description in the figure legend (l916ff). Eg5 is degraded by the APC/C at the G1 phase (Eguren et al., Cell Rep. 2014) and S10 on histone H3 is phosphorylated at the end of G2 phase and trough-out mitosis. To better define mitosis (and the end of M) the authors should also blot for cyclin B1 and/or cyclin A2. The choice of Eg5 and Wee1 is not ideal as their degradation profile is not as sharp and poorly visible in the data presented. Furthermore, it would benefit their comparison between different cell lines if in both experiments the same markers are analysed. Finally, the pS10 blotting and the FACS profiles in Figure 5 indicate that the cells are still significantly enriched in G2 or M phase as the pS10 peak is still very high by the end of the experiment. This indicates that THP-1 cells have a longer cell cycle than RPE-1 cells or that the release in THP-1 is slower than in RPE-1 cells at a certain cell cycle stage. Can the authors comment on this and better describe their data?

-The authors should include a table with all 25 cell lines tested indicating, which cell lines are suitable for CDK4 inhibitor based synchronization and which are not. They mention in the text only few non-responsive ones, but this information is important for the reproducibility of their data and will save the time of others applying this approach to non-responsive cells. Also, the identity of 5th cell line that responded well to CDK4i synchronization is not given.

[TEST] Reviewer 1: Release from the cell cycle arrest with Cdk4/6 inhibitors generates highly synchronised cell cycle progression in human cell culture by Trotter and Hagan

In this very interesting manuscript Trotter and Hagan use Cdk4/6 inhibitors, predominately the drug palbociclib, to arrest hTERT-RPE1 cells close to 100% in G1 phase of the cell cycle. Wash out of the drug then triggers synchronized progression of the cells through the cell cycle. The authors carefully define the conditions that allow high efficient synchronisation with palbociclib and establish that this arrest and release protocol does not cause DNA damage as this is the case for the conventional thymidine block and release scheme. The synchronisation protocol described by Trotter and Hagan will help many researchers to perform highly reproducible and meaningful cell cycle analysis experiments in cell lines that before resisted the conventional protocols. I therefore strongly support publishing of this manuscript in Open Biology. I have only minor points that the authors may want to address before publication.

Specific points

1. Line 276: “indicated by black and red line sin the FACS plots”.
2. Lines 291-295: complicated long sentence. Please simplify.
3. Line 294; remove “data”.
4. Line 300: “open black squares”.
5. Fig. 7d: why do authors use grey and blue colours? I guess that two synchronized cultures were used. The colour code on top should follow the colour code in the bars: light, medium, high. The colour code is not completely clear. I guess EdU means EdU positive (+/- gamma-H2AX) and gamma-H2AX (+/- EdU). Please clarify.
6. An alternative way of analysing Fig. 7d is counting G2 cells (CENP-F positive) for gamma-H2AX staining with and without synchronization. The synchronized cells would be analysed after 14-16 h. This would address whether the synchronisation scheme leads to cells with DNA damage.
7. The authors should explain better how the experiment in Fig. 9 was done. I guess that cells were incubated with the given palbociclib concentrations for the stated time. The medium was then changed (no palbociclib + nocodazole) and cells were incubated for 24 h followed by FACS analysis. How do the authors explain the behaviour of A549? Shorter incubation