1,068 Matching Annotations
  1. Last 7 days
    1. Gracechurch Street

      Gracechurch Street is a main road in London and is historic for its shops and restaurants and serves as an entrance to Londons Leadenhall Market which has been open since the 14th century. https://janeaustensworld.wordpress.com/tag/gracechurch-street/

    1. Menu items are links <a>, not buttons. There are several benefits, for instance: Many people like to use “right click” – “open in a new window”. If we use <button> or <span>, that doesn’t work. Search engines follow <a href="..."> links while indexing.

      key point on why use 'a' tag instead of buttons for navigation purposes

    1. They’ve learned, and that’s more dangerous than caring, because that means they’re rationally pricing these harms. The day that 20% of consumers put a price tag on privacy, freemium is over and privacy is back.

      Google want you to say yes, not because they're inviting positivity more than ever, but because they want you to purchase things and make them richer. This is the essence of capitalism.

    1. Suppose college tuition was free and every first-year had a guaranteed job lined up for after graduation. This parallel universe does exist at military-service academies—and at West Point, Annapolis, and Colorado Springs, humanities majors are at about the same level as they were in 2008.

      Huge clue re: precarity's effect. Also a bit of a portent in the possible effect of a Universal Basic Income. Which has to now become a new tag I use...

  2. May 2019
    1. yes there are many threads

      Yes. Every time one of these accounts appears, I tag it as a throwaway account. I am doing that will all immediately registered accounts in those threads, no matter who they look like. This allows later analysis, because, without this, the accounts become [deleted] and cannot then be correlated. Interesting patterns appear when this is done. Sometimes it becomes possible to correlate accounts across multiple platforms.

    1. vector, under the phage T7 promoter, in BL21 (DE3) cells, and under the T5 phage promoter, in the pQE30 vector for expression in SG13009[pREP4] and M15[pREP4] cell strains. For cloning in pRSET B, the full length bZP3 initially subcloned in the pBacPAK8 vector at the Kpn I and Sac I sites was released after digestion with Kpn I and EcoR I and cloned in a similarly restricted pRSETB vector inframe with an N-terminal His6 tag. For cloning in the pQE30 vector, the pBacPAK8 carrying the full length bZP3 was initially digested with Not I, filled in with Klenow and then digested with Kpn I. The purified bZP3 fragment was then cloned in the vector digested with Kpn I and Sma I in frame with an N-terminal His6 tag. Though transformants positive for the bZP3 insert in the right reading frame were recovered, no expression could be detected by SDS-PAGE or immunoblots in either case. An alternate strategy was then devised in which an internal fragment of the gene, excluding the signal sequence and the transmembrane-like domain, following the putative furin cleavage site, was amplified by PCR using the forward primer 5'-CGGGATCCCAACCCTTCTGGCTCTTG-3' incorporating a BamH I site and the reverse primer 5'-CCGAGCTCAGAAGCAGACCTGGACCA-3' incorporating a Sac I site. The PCR was done in a 50 J!l volume using 50 pM of each primer and Vent polymerase for extension. The pBluescript-bZP3 (1 0 ng) having a full length bZP3 insert was used as the template and was initially denatured at 95°C for 10 min. Amplification was carried out for 35 cycles of denaturation at 95°C for 2 min, primer annealing at 600C for 2 min and extension at 72°C for 3 min followed by a final extension at 72oc .for 15 min. The amplified bZP3 fragment was digested with BamH I and Sac I and cloned in frame downstream of a His6 tag under the T5 promoter-lac operator control in the pQE30 vector. The authenticity of the construct was confirmed by N-terminal sequencing using an upstream sequencing primer GGCGT ATCACGAGGCCCTTTCG.
    1. carriage was sent to meet them at — , and they were to return in it by dinner-time.

      Hackneys, or public carriages for hire, made their first significant appearance in the early 17th century. By 1694, this method of transportation was very popular so the Hackney Coach Commission was established in London.

      https://janeaustensworld.wordpress.com/tag/regency-transportation/

    2. dressing-room

      A room primarily used during one's morning routine for dressing and washing. A woman's dressing room was made to be private and comfortable, and the intimacy of these small places allowed women to entertain small parties of other female guests. The wealthier the woman, the more luxurious her dressing room.

      https://janeaustensworld.wordpress.com/tag/18th-century-dressing-rooms/

    1. argP+, argPd-S94L, argPd-P108S, argPd-P274Sfragment downstream of the phage T7-promoter, such that the encoded proteins beara C-terminal His6-tag provided by the vector DNA sequence. Theresultant plasmid was transformed into strain BL21(DE3) which has the T7 RNA Polymerase under the isopropyl thio-β-D-galactoside (IPTG) inducible lacUV5promoter.The resultant strains were grownin LB (500-1000 ml) to an A600of around 0.6and were then induced with 1 mM IPTG and harvested after 4-hrs of induction.Bacterial cells were recovered by centrifugation, resuspended in 20 ml of lysis buffer(20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mM DTT and 10 mM imidazole) containing20 μg/ml lysozyme, and lysed by sonication with 30-sec pulses for 10-min. Theprotocol for His6-ArgP(ArgPds)protein purification involved (i) passing the lysate through a 5ml Ni-NTA (Qiagen) chromatographic columnequilibrated with lysis buffer, (ii) washing thecolumn with 100 ml of washing buffer (20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mMDTT; 30 mM imidazole), and (iii) elution of His6-ArgP(ArgPds)from the column with elutionbuffer (20 mM Tris-Cl, pH-8;300 mM NaCl; 10 mM DTT and 250 mM imidazole) andcollection of 1.5 ml eluate fractions (10 fractions). The fractions were tested forprotein by Bradford method and the protein-carrying fractions (generally tubes 2 to 5)were pooled and dialysed in a 1:200 volume ratio against 20 mM Tris-Cl, pH-8 with 10mM DTT, 300 mMNaCl for 5 hrs followedby a change to buffer of composition 20 mM Tris-Cl, pH-8 with 10 mM DTT, 300 mM NaCl and 40% glycerol for 24 hrs. The proteins were concentrated by centrifugation toaround 1 mg/ml by using Amicon filter (pore size 10-KDa) and stored at −20ºC or −70ºC
    1. The TEI's adoption as a model in digital library projects raised some interesting issues about the whole philosophy of the TEI, which had been designed mostly by scholars who wanted to be as flexible as possible. Any TEI tag can be redefined and tags can be added where appropriate

      Question - What were some of the issues that arose with the TEI's adoption as a model in digital library projects?

      The ability to add tags where needed throughout the texts seems to be a positive aspect due to the added ease of searching key words and it being included in said search.

  3. shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
    1. After 2-3 h exposure,phosphorimagerscreenwas scannedon Fuji FLA-9000 to acquire hybridization images. Next,signal intensity for each spot on the membrane for both input and outputsampleswas quantifiedusing Fuji Multi Gauge V3.0 software andpercentage intensity foreach spot relative tothe whole signal intensity ofthe membranewas determined.To identify mutants with altered survival profiles,ratio of output (Op) to input (Ip) signal for each spot (oligonucleotide tag)present on the membranewas calculated.Mutantsdisplaying at least 6-fold higher and 10-fold lower survival were selectedas “up’ (Op/Ip= 6.0) and ‘down’ (Op/Ip = 0.1) mutants, respectively
    2. 24 h post infection, THP-1 macrophages were washed thrice with PBS, lysed in water and recovered yeast cells were used to infect THP-1 cells at a MOIof 1:10. Three rounds of macrophage infection foreach mutant pool were carried out to enrich for the desired mutants in the final population. The lysate of 3rdround infection was inoculated in YPD medium for overnight (output). Cells were harvested, genomic DNA isolated from each input and output cell pellet andunique signature tags were PCR-amplified with P32-labeledα-dCTP using primers complementary to theinvariant region flanking each unique tag sequence. LabeledPCR products were denatured at 95°C for 10 min, chilled on ice and were hybridized tonylon membranescarrying immobilized plasmid DNA containing 96 unique tagsfor 14-16 h at 42°C.Membranes were washed twicewith 0.1X SSC bufferand exposed to phosphorimager screen for 2-4 h. Radioactive counts for each spot were quantified using Image Quant and Fuji Multi Gauge V3.0 software. Relative percentage intensity for individual spot was calculated with respect to allspots present oneach hybridizedmembrane
    3. YPD-grown cultures (0.05 OD600) of each mutant pool (96 mutants, each carrying a unique signature tag) were either inoculated in YPD medium for overnight (input) or used to infect differentiated THP-1 cells (1X106). After 2 h incubation, non-cell-associated yeastcellswere removed by washing THP-1 cellsthricewith PBS. At
    1. Afteractivating Hypothes.is in the browser, a learner can annotate any piece of text on a webpage with her own ideas, to whichother learners could respond (Principle 1). Tags can be attached to a Hypothes.is annotation, enabling the aggregation of webannotations that are scattered across webpages through a tag (Principle 2). Because Hypothes.is adheres to the Open AnnotationModel, annotations are uniquely identifiable and retrievable, allowing portability of ideas not widely supported in threadeddiscussion forums (Principle 2). The search functionality of Hypothes.is offers means to aggregate annotations according to avariety of criteria such as by tags, users, user groups, and annotated web URLs. Users are thus able to enter the discourse notonly from a specific webpage (e.g., the textbook), but also from the search page that provides another view of the discourse(Principle 3)

      Alignment statement for Hypo

    1. The following antibodies were used in the present study:Primary antibodies against GAPDH (anti-rabbit), FLAG (anti-mouse), Immunoglobulin (IgG, anti-rabbit or anti-mouse),profilin-1 (anti-rabbit), tubulin (anti-mouse) and ubiquitin (anti-rabbit) were obtained from Sigma Aldrich Chemicals(St Louis, MO, USA). Antibodies againstAKT (anti-rabbit), cleaved caspases-3, 8 and 9 (anti-rabbit),HA-tag(anti-rabbit), Myc-tag (anti-rabbit), p21 (anti-rabbit), phospho-p53 (anti-mouse), PTEN (anti-mouse), phospho-AKT (Ser473; anti-rabbit), phospho-GSK-3β (Ser9; anti-rabbit), phospho-IKKα/β (Ser177/181; anti-rabbit), phospho-IκBα (Ser32; anti-rabbit), and phospho-p65 (Ser276; anti-rabbit) were obtained from Cell Signaling Technologies(Danvers, MA, USA), whereas antibodies for cox-2 (anti rabbit), c-Rel (anti-rabbit), ICAM-1 (anti-rabbit), IKKα/β (anti rabbit), IκBα (anti-rabbit), Mdm2 (anti-rabbit), PARP-1/2 (anti-rabbit), Rel-B (anti-rabbit), p50 (anti-rabbit), p53 (anti-mouse), p65 (anti-rabbit) were obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA).HRP (Horse radish peroxidase)-conjugated secondary antibodies (anti mouse and anti-rabbit) were obtained from Bangalore Genie(Peenya, India). For immuno-fluorescencestudies, secondary antibodiesconjugated toAlexa Fluor (488 and 594, anti-mouse and anti-rabbit) were obtained from Molecular Probes, Invitrogen(Eugene, OR, USA)
    1. immunoprecipitation by using substrate specific antibody or pull-down by affinity trapping the substrate tag. The IP/pull-down complexes wereanalyzed by detecting the ubiquitination of substrate protein by using either substrate specificantibody or ubiquitin antibody through western blotting
    2. using gateway cloning method (Invitrogen). P73domain deletions were cloned in SFB destination vector. WWP2, WWP1, HACE1, E6AP, and PPM1G were cloned into SFB (S-protein/Flag/streptavidin binding protein (SBP) triple tag), GFP,and Myc mammalian destination vectors using the Gateway cloning technology (Invitrogen). WWP2 domain deletions were cloned into Myc-destination vector. WWP1 domain deletions were cloned into SFB-destination vector. PPM1G domain deletions were cloned into SFB mammalian destination vector using Gateway cloning. Bacterially expressing GST-p73, GST-∆Np73, GST-PPM1G, MBP-WWP1, MBP-WWP2, GST-WWP2, GST-WWP1 and GST-HACE1 were generated by using gateway technology. Ubiquitin WT and all the mutants were cloned into hemagglutinin (HA) mammalian destination vector. Flag-tagged Dvl2was purchased from Addgene. Dvl2 domain deletions were cloned into SFB-destination vectors. All the plasmid constructs generated in the present study are mentioned in table 2.Table 2: Plasmids used in the study
    1. ability to take notes anchored to specific passages of text, and have them tagged and searchable across papers, are such major benefits

      For this to happen, start with:

      1. Open an article in the web browser (Google Chrome or Firefox) that can support pdf.js or epub.js or html if that is available
      2. Open hypothesis app, and start annotating at passages (as in this annotation)
      3. Add a consistent tag (see the other how_to tagged articles)
      4. Then search all related hypothesis annotations and notes on pages using those tags and organise knowledge that way.
    1. agree on a standard set of tags to classify sets of resources

      This is step one. Set up a standard set of tags. These tags will then classify resources. Let's say I want to classify all studies on polygenic risk scores on the web. These documents come in the form of HTML, or PDF. If on the top of that, you'd also like to tag something like tutorial, or software to do the job, then write those tags as well when you come across a resource and tag it. So, a list of tags to learn about polygenic risk scores could be something like:

      • polygenic risk score
      • polygenic_risk_score
      • tutorial
      • utility
      • why_how So, all resources that pertain to polygenic risk scores can now be divided into a set of main document types: some that discuss tutorials and how tos, others discuss the utilisation values and debates. Later, these could be reassembled.
  4. Apr 2019
    1. seminary

      Austen is referring to a boarding school attended by young women from wealthy families who, for some reason, were not educated at home by a governess. https://judeknightauthor.com/tag/girls-education-in-regency-england/

    2. any complaint which asses' milk could possibly relieve

      Donkey milk was considered a viable medical treatment from antiquity (Cleopatra bathed in it) until the turn of the 19th century, when it largely went out of fashion. It was considered a generic cure for a variety of conditions, including gout, scurvy, coughs, colds and asthma. For many, donkey milk caused stomach problems and "lactose intolerance."

      https://georgianera.wordpress.com/tag/asses-milk/

    1. Annotation Profile Follow learners as they bookmark content, highlight selected text, and tag digital resources. Analyze annotations to better assess learner engagement, comprehension and satisfaction with the materials assigned.

      There is already a Caliper profile for "annotation." Do we have any suggestions about the model?

    1. 1) We identify and investigate two major kinds of lin-guistic activities on Quora: user level [e.g., basicactivities like posting a question/answer/comment aswell as linguistic styles that involves word/char usage,and part-of-speech (POS) tag usage] and questionlevel [e.g., content, topic associations, and edits for aquestion). Remarkably, many of these activities arefound to have a natural correspondence to the qualitiesthat human judges would consider while deciding if aquestion would remain unanswered (see Table I for a setof motivating examples).2) We perform an extensive measurement study to showthat answerability can be indeed characterized based onthe above-mentioned linguistic activities.3) A central finding is that the language use patterns ofthe users is one of the most effective mechanisms tocharacterize answerability.

    Annotators

    1. We identify and investigate two major kinds of lin- guistic activities on Quora: user level [e.g., basic

      activities like posting a question/answer/comment as well as linguistic styles that involves word/char usage, and part-of-speech (POS) tag usage] and question level [e.g., content, topic associations, and edits for a question). Remarkably, many of these activities are found to have a natural correspondence to the qualities that human judges would consider while deciding if a question would remain unanswered (see Table I for a set of motivating examples). 2) We perform an extensive measurement study to show that answerability can be indeed characterized based on the above-mentioned linguistic activities. 3) A central finding is that the language use patterns of the users is one of the most effective mechanisms to characterize answerability.

    1. appears

      The link is to my blog, but is defective. (a link that works is this, but this is to all occurrences of a tag.) Elsewhere, I found the page that the author was attempting to cite. Here I am not. The text is confused. I have never seen Oliver write "under his brother's name," but he has mostly claimed his brother is different, and then he claimed that he was lying about that and had been lying for years, then he took it back. Liars lie, no way around it. If a liar says "I'm a liar," a sane answer is "Not always." After all, stopped clock.

    2. claim he made this up.

      This is a link to my blog, the URL is broken, though. The core points to this, a tag. Searching that display for "schizo," I find eventually material copied from this page: Authentic Darryl Smith on himself That page is Darryl making the claim of "made up." Recently, socks on Reddit -- probably also Darryl -- have repeated the claim that Oliver denied "schizophrenia," but I have never seen Oliver openly deny it. And it makes sense.

    1. If this is a production situation, and security and stability are important, then just "convenience" is likely not the best deciding factor (any more than leaving your house unlocked all the time might be "convenient").

      如果这是生产情况,安全性和稳定性很重要,那么“便利”可能不是最好的决定因素(不仅仅是让你的房子一直解锁可能是“方便的”)

      • 您可以考虑将每个push to registry的版本 - 以某种形式(毕竟,您发布了新版本的代码,并使其他人可以访问)。
      • :latest与Git存储库中的master分支相当。是否每个push to master都考虑准备投入生产?
      • Releases将(通常)通过验证过程(CI/QA /acceptance/etc)。是否应首先验证master中的更改,并且仅在验证(标记并)部署到生产之后?
      • 发行版(Releases)带有版本;这可以是显式版本(标记),也可以是隐式(不可变标记:图像的摘要)

      显式版本 -- image tag<br> 隐式版本 -- 不可变标记 :image digest

    2. However, there is not a 1:1 relation of digests to tags, so when pulling an image by digest, only the digest is known. If you happen to have an image pulled (manually) with a tag that matches that digest, the tag is shown, but not otherwise

      但是,摘要与标签之间没有1:1的关系,因此在通过摘要pull image时,只知道摘要。如果您碰巧使用与该摘要匹配的标记(手动)拉出图像,则会显示标记,否则不会显示

    1. Impact of the Legion 9 plain 2019-04-16T02:18:41+00:00 Joseph Brown 5eac7ef3705d9f80e567b77e809a27064bd00249 1 2019-04-11T22:00:35+00:00 Joseph Brown 5eac7ef3705d9f80e567b77e809a27064bd00249 Organization of the Legion

      As far as I can tell, you are using the tag function for the purpose of forward and backward movement. I'd reconsider using it for this purpose.

    1. “the expression or application of human creative skill and imagination […] producing works to be appreciated primarily for their beauty or emotional power.”

      Who is the speaker on this quote? Braidotti? I would tag the source or do what you did above (Author last name, page #)

    1. Alexander McQueen: Relationality between human and non-human Charles Sirisawat-Larouche 6 plain 2019-04-10T17:03:44+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Contents of this tag: 1 2019-04-04T13:19:50+00:00 Deterritorialize the Fashion Practices 11 vistag 2019-04-10T17:15:57+00:00 1 2019-04-01T13:48:01+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 The Deconstructivist Approach Charles Sirisawat-Larouche 22 plain 2019-04-10T16:51:05+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Contents of this tag: 1 2019-04-10T11:43:52+00:00 Alexander McQueen: Relationality between human and non-human 6 plain 2019-04-10T17:03:44+00:00 1 2019-04-04T13:19:50+00:00 Deterritorialize the Fashion Practices 11 vistag 2019-04-10T17:15:57+00:00 1 2019-04-10T13:46:55+00:00 The display of its fashion is done toward alien like figure or cyborg incarnation which allow a certain extent of dis-identification. 3 plain 2019-04-10T13:48:27+00:00 1 2019-04-10T12:26:35+00:00 Fashion and Anti-fashion Dichotomy 2 plain 2019-04-10T16:34:01+00:00 1 2019-04-10T12:22:36+00:00 Phallogocentrism 3 plain 2019-04-10T16:38:25+00:00 1 2019-04-10T12:26:06+00:00 Self-Styling and Self-Fashion 3 plain 2019-04-10T16:39:08+00:00 1 media/Fashion-Looks-Forward_Utopian-Bodies_Exhibition_Stockholm-_dezeen_936_12.jpg 2019-04-01T15:44:50+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Post-Gender Charles Sirisawat-Larouche 15 visual_path 2019-04-10T17:26:43+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Contents of this tag: 1 2019-04-04T13:19:50+00:00 Deterritorialize the Fashion Practices 11 vistag 2019-04-10T17:15:57+00:00 1 2019-04-10T00:30:04+00:00 Rei Kawakubo and the excessive use of material as a strategy to reassert the conceptual embodied entity toward subversive site. 5 Rei Kawakubo and the excessive use of material as a strategy to reassert the conceptual embodied entity toward subversive site. Click and Drag to Annotate plain 2019-04-10T16:17:23+00:00 1 media/IMG_5473 2.jpg media/Animated GIF-original.mp4 2019-04-01T13:55:26+00:00 Thierry Mugler: The contradictory abjection 27 visual_path 2019-04-10T17:32:51+00:00 1 media/2000px-Polka_dots.svg.png media/Animated GIF-original.mp4 2019-04-04T13:16:11+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Posthuman Museum Practices: From Runway to exhibition Charles Sirisawat-Larouche 34 Preceding the essence of fashion tags 2019-04-10T17:47:08+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Contents of this tag: 1 2019-04-04T13:19:50+00:00 Deterritorialize the Fashion Practices 11 vistag 2019-04-10T17:15:57+00:00 1 2019-04-08T15:25:59+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Self-organizing: Curator, Curatorial. Curating... Charles Sirisawat-Larouche 10 visual_path 2019-04-10T14:09:14+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Contents of this tag: 1 2019-04-04T13:19:50+00:00 Deterritorialize the Fashion Practices 11 vistag 2019-04-10T17:15:57+00:00 1 media/IMG_5473 2.jpg media/Animated GIF-original.mp4 2019-04-01T13:55:26+00:00 Charles Sirisawat-Larouche b6ad6d1a6de4c926cdef3dafe6adb7d5f4702fd8 Thierry Mugler: The contradictory abjection

      Nice idea to use tags. Relevant to the style of assignment.

    1. Can we at this point have the option of also adding a "TAG" function It's much faster to be able to tag as when new questions are being inputted

      Also if the question TAG is shown here, new quizzes can be created on the spot with a specific question TAG (if that makes sense)

    1. we injected Cre recombinase–inducible adeno-associated viruses (AAV) expressing the optogenetic channelrhodopsin-like ChIEF fused with a tdTomato reporter [AAVdj-CAG-DIO-ChIEF-tdTomato (driven by the CAG promoter) (10, 11)] bilaterally into the rostral ZI of vesicular GABA transporter (VGAT)–Cre mice that express Cre recombinase in GABA neurons

      To target a neuron population of interest, eg. those that express GABA, scientists use genetically modified viruses (AAVs) to deliver proteins into the brain (such as optogenetic tools).

      This is achieved by using two tools: 1) a mouse line that expressed the enzyme Cre recombinase in a specific population of neurons (eg. those that express the GABA transporter VGAT). 2) an AAV that expresses an optogenetic protein only in the presence of Cre. The AAV is injected into the brain region of interest in the Cre mice. This AAV has a tdTomato tag which allows the injection site to be visualized under a fluorescent microscope.

      For further information on these tools see: Optogenetics- YouTube

      The ZI in both hemispheres of the brain was injected with the AAV (bilaterally), with the region lying towards the front of the brain (rostral) being targeted. The optogenetic tool used (ChIEF) activates neurons when blue light is shone on the cells.

    1. Safety advocates urged college students to match the vehicle color and model, the license tag number, and the photo of their ride-share drivers before getting in a vehicle and make the driver say their names to them before they introduce themselves.

      Students should follow this to be safe.

    2. Safety advocates urged college students to match the vehicle color and model, the license tag number, and the photo of their ride-share drivers before getting in a vehicle and make the driver say their names to them before they introduce themselves.

      Always make sure to do this

    1. But the offer came with a hefty price tag, the negotiators said, which the system was not willing to pay.

      Doesn't it seem like everything comes with a hefty price these days...

    1. Dr. Sanha Kim should be moved over to Columbia. We need to add a space holder for Cynthia Loveland under Columbia doctors. For all doctors except Tanya Tag, list them as 'Emergency Veterinarian' instead of emergency and critical care. Under 'doctors' Dr. Tag looks great, could you add Towson & Columbia next to Medical Director? For Dr. Tag under administration, remove her middle initial and change DVM to match DACVECC as displayed for her under doctors. Leave medical director Towson & Columbia. Please display leadership in this order: Angelina Morgan, Tanya Tag, Tamara Santesse, Elizabeth Moyers, Beth Faux, Ashley Carlson, Victoria Syczurowski, Jeff Hawkins, Christine Wolff, Paul Heggie, Joey Sumby (I spelled his name wrong last time)

      Beth Faux should display as "Beth Faux, RVT, VTS (ECC). Her title should be listed as Technician Manager Columbia. Tamara Santesse's title should be Office Manager, Towson. Elizabeth Moyers' title should be Office Manager, Columbia Victoria and Ashley's title are: Technician Co-Manager Towson Jeff Hawkins' title should be Financial Manager Towson & Columbia Paul Heggie's title should be Facilities Manager Towson & Columbia Joey Sumby's title should be Facilities Supervisor Columbia

      Love the super heroes-are there gender specific?

  5. Mar 2019
    1. Twitter Facebook Instagram Impressum Haftungsausschluss

      Alignierung kann angepasst werden

      • gutes Teaserbild. Allerdings nimmt zuviel Platz. Für den Hauptinhalt (Wanderungen) muss man sofot scrollen. Eine Alternativ wäre mit einem "Karrussell" von den letzten 3 Wanderungsbilder oder so.
    1. Behaviorism

      Learning-Theories.com published a very handy few pages that describe various learning theories. This is a quick, straightforward, simple way to access information on the different theories. This article, Behaviorism, explains that the theory assumes learners learn by responding to external stimuli in their environment. Learning under behaviorism is characterized by a change in the learner's behavior. I use this in my horse training as I use both positive reinforcement (clicker training) and negative reinforcement (pressure-release) to structure my horse's behaviors. Behaviorism can be translated to human work, too. I've used TAG teaching (clicker training for humans) to teach people to get on and off horses with ease and also to trim horses' hooves. I also use it to clicker train my cats! 6/10

    1. Republicans immediately painted the plan as “disastrous,” pointing to studies that suggest the price tag could be as high as $32 trillion.

      Source?<br> Which Republicans?

  6. digitalempathyvet.com digitalempathyvet.com
    1. health exam and vaccinations - $167.53 includes Doctor exam, rabies, Dhlpp, bordetella, send out to lab fecal, and heart worm occult and parish tag. puppy care 1st shots= $137.40 2nd- 9 weeks is $143.29 3rd - 12 weeks $147.01 4th - 15 weeks $187.08 Micro chips- $63.13 feline neuter- $100.89 feline front declaw- $232.87

    1. disallows it in our Public channel and in groups

      You can, however, add a tag to a highlight in order to share it to the Public channel or a group.

    1. personalize learning infographic

      This is not quite what it sounds like. It is a Pinterest style page with links to assorted articles that relate to personalized learning, most of which are presented in an infographic. It is sufficiently useful if one has the patience to click through to the infographics. Usability is satisfactory although the top half of the page is taken up with graphics that are not directly related to the content. rating 3/5

    1. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 1 DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5 Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, Masahisa Katsuno Review timeline: Submission date: 1 October 2017 Editorial Decision: 13 November 2017 Revision received: 27 February 2018 Editorial Decision: 6 April 2018 Revision received: 4 May 2018 Extra communication: 17 May 2018 Editorial Decision: 12 June 2018 Revision received: 7 January 2019 Editorial Decision: 11 Jannuary 2019 Revision received: 1 March 2019 Accepted: 1 March 2019 Editor: Céline Carret Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 13 November 2017 Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the two referees whom we asked to evaluate your manuscript. As you will see from the reports below, the referees find the data intriguing. Unfortunately, at this stage the work needs to be considerably strengthen to be more conclusive, and both referees provide good suggestions for that. Additional controls and experiments and adding mechanistic understanding seem to be required for the paper to be further evaluated in EMBO Molecular Medicine. Given that the referees find the message interesting, we would be willing to consider a revised manuscript with the understanding that the referee concerns must be fully addressed and that acceptance of the manuscript would entail a second round of review. I should remind you that EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. I realize that addressing the referees' comments in full would involve a lot of additional experimental work and resources and I am uncertain whether you will be able (or willing) to return a revised manuscript within the 3 months deadline. I would also understand your decision if you chose to rather seek rapid publication elsewhere at this stage. I look forward to seeing a revised form of your manuscript as soon as possible. Please read below for important editorial formatting and consult our author's guidelines for proper formatting of your revised article for EMBO Molecular Medicine. Should you find that the requested revisions are not feasible within the constraints outlined here and choose, therefore, to submit your paper elsewhere, we would welcome a message to this effect. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 2 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): Better control should be used as detailed to Authors Referee #1 (Remarks for Author): The study by Kondo al coll. Reports that the toxicity of the mutant AR responsible for SBMA could be ascribed to altered DNA methylation, and that this alteration could be reverted by the inhibition of this mechanisms using DNA methylation inhibitor such as RG108. In addition, the Authors have demonstrated that the hyper-methylation of the Hes5 gene correlates with the AR-97Q toxicity and that overexpression of Hes5 rescues from SBMA phenotype in cell models of the disease. While the study is potentially interesting, several points must be addressed in order to definitely prove that DNA methylation is relevant in SBMA. Major points:

      1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice.
      2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses.
      3. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motorneurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT.
      4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript
      5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells?
      6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108 EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 3
      7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers.
      8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice?
      9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR- 24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Minor Points
      10. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR.
      11. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Referee #2 (Comments on Novelty/Model System for Author): See comments to authors for 1 and 4. One issue is that validation in the NSC transformed cell line model is not sufficient. Referee #2 (Remarks for Author): In the Kondo et al. paper, a role for altered methylation in the polyglutamine disease SBMA is sought. The authors show Dnmt1 is upregulated within spinal motor neurons and suppression by siRNA or by a chemical inhibitor RG108 can suppress neuronal cell death. The authors translated the inhibitor into a pre-clinical mouse model and found evidence for suppression of SBMA neurodegenerative phenotypes. Intriguingly, this occurred downstream of polyglutamine aggregation, as aggregation was not affected. The authors show that cell viability in a NSC cell model of SBMA enabled by RG108 treatment depends on Hes5 expression, and that re-expression of Hes5 is sufficient to rescue viability. While the topic of methylation in neurodegeneration has not been addressed much, making the work intriguing, there are a number of problems with the investigation that undermine one's confidence in the results. The authors' explanation for which genes are affected by altered methylation is very superficial, and what Hes5 is doing is entirely unclear. As the validation of Hes5 takes place in only the transformed cell line model of SBMA, it seems rather premature to implicate Hes5 without providing a more thorough understanding of what it is doing. More concrete data is needed to balance what appears at times as speculation. Specific concerns that should be addressed to strengthen the manuscript are as follows: 1) The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. 2) A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and reviewers) can determine if the conclusions are valid. The problematic Figures are as follows:
      12. Fig 1C
      13. Fig 1D
      14. Fig 1E, EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 4
      15. Fig 1F
      16. Fig 2D
      17. Fig 3H
      18. Fig 5H
      19. Fig 6F
      20. Fig S1
      21. Fig S2
      22. Fig S3
      23. Fig S4
      24. Fig S5 3) All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. 4) As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Minor points: -There remain quite a few grammatical errors and typos, the authors should review the manuscript for these -The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? -Can the authors kindly provide the rationale for choosing RG108? -Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. -Figures 3A-D are missing significance labels -Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. 1st Revision - authors' response 27 February 2018 Referee #1: Major points: Point 1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice. Response 1: We thank the Referee for this important suggestion. In response to the proposal, we performed RT-qPCR of Dnmt1, Dnmt3a, and Dnmt3b using spinal cord samples from each group of mice (Fig. 1C). This analysis revealed that Dnmt1 mRNA level was up-regulated in AR-97Q mice as well as protein level. Point 2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 5 Response 2: In response to the Referee’s comment, we added the data of AR-24Q mice in Fig. 1A, 1B, 1C, 1D, 1E, 5I, and 5J, so that we excluded the influence of normal AR to the protein level and mRNA level of Dnmt1, Dnmt3a, Dnmt3b, and Hes5. Point 3-1. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. Response 3-1: We changed the term “expression” to “protein level” or “level” for the part of manuscript describing western blotting. In order to evaluate the gene expression level of Dnmt1, we performed RT-qPCR of NSC24Q and NSC97Q cells, and confirmed that Dnmt1 mRNA is expressed at a higher level in NSC97Q compared with NSC24Q (Fig. 2B). Point 3-2. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motor neurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. Response 3-2: We changed the sentence according to Referee’s suggestion (Page 7, Line 1-2). Point 3-3. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT. Response 3-3: To address the comment, we added the data comparing protein levels of Dnmts and Dnmt1 mRNA level in NSC97Q with or without DHT treatment (Fig. 2C and D). Moreover, we analyzed the influence of Dnmts knockdown on the cell viability of DHT-untreatd NSC97Q cells (Fig. 2F). Furthermore we evaluate the effect of RG108 to NSC97Q cells without DHT (Fig. 2I). The results of these additional experiments showed that Dnmt1 is not up-regulated in DHTuntreated NSC97Q cells, and that neither siRNA-mediated knockdown of Dnmts nor RG108 treatment alters the cell viability of NSC97Q cells without DHT treatment. Point 4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript. Response 4: According the Referee’s suggestion, we added the data of WST-8 assay of DHTtreated NSC24Q cells with siRNA-mediated knockdown of Dnmts (Supplementary Fig. 9) and RG108 treatment to DHT-treated NSC24Q (Fig. 2H and J). Both knockdown of Dnmts and RG108 had no effect on the cell viability of NSC24Q with DHT treatment. Point 5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells? Response 5: The DNA methylation array analysis we utilized was developed for human materials, but could not be applied to mouse samples at the time we performed experiments. Therefore we analyzed the samples of SH-SY5Y, human neuronal cells. In order to confirm that the phenomena observed in human cells are reproduced in murine cells, we performed all the experiments using cell samples derived from SH-SY5Y (Fig. 5 A, 5B, 5C, 5D, 5F, 5G, and 5H; Supplementary Fig. 12; Fig. 6A and 6B) and those from NSC34 (Fig. 2A and 2C; Supplementary Fig.13 and Fig. 6 C and 6D) in parallel. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 6 Point 6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108. Response 6: Our preliminary experiments revealed that Dnmt1 protein level was already elevated in the spinal cord of 6-week-old AR-97Q mouse (Figure for Referee shown below). This is the reason why we decided to start the brain injection treatment with RG108 to 6-week-old age mice. Point 7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers. Response 7: We changed the sentence to “DNA methylation array analysis using these cells revealed that DNA methylation of CpG islands is intensified in several genes. However, total DNA methylation level was not altered between SH24Q and SH97Q (Supplementary Fig. 11).” (Page 9, Line 7-9) Point 8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice? Response 8: To address this issue, we performed western blot using nuclear and cytoplasmic fraction of spinal cord lysate obtained from wild-type and AR97Q mice. Both in wild-type and AR97Q mice, Hes5 showed cytoplasm-dominant localization (Figure for Referee shown below). Although we cannot exclude the possibility that nuclear localization of Hes5 is somehow impaired in AR97Q mice, the western blot indicates that Hes5 is down-regulated both in nucleus and cytoplasm in SBMA model mouse, presumably resulting in the faint nuclear staining in the immunohistochemistry of the AR97Q mice. Point 9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR-24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Response 9: According to the comment, we added the data comparing DHT(-) and DHT(+) as shown in the Table for Referee shown below. As AR aggregation is not detectable in NSC97Q cell without DHT treatment, we did not perform the counterpart experiment for Fig. 7G and H. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 7 The data Referee pointed The corresponding data in the revise version Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig6. B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Minor Points Point 1. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR. Response: We are thankful to the suggestion. We changed RT-PCR to RT-qPCR throughout the manuscript. Point 2. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Response: We changed the term from “Wild” to “Wild-type”. Referee #2 Point 1. The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. Response 1: We thank the Referee for pointing this important issue. We now confirmed that relative mRNA level of human AR was not altered by RG108 treatment (Fig. 4E). This data indicated that RG108 had does not suppress AR transgene expression. Point 2. A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and Referees) can determine if the conclusions are valid. The problematic Figures are as follows:
      25. Fig 1C
      26. Fig 1D
      27. Fig 1E,
      28. Fig 1F
      29. Fig 2D
      30. Fig 3H
      31. Fig 5H
      32. Fig 6F
      33. Fig S1
      34. Fig S2
      35. Fig S3
      36. Fig S4
      37. Fig S5 Response 2: In response to the comment, we quantified the data as described below. Furthermore, we described the number of samples analyzed for quantitation in the figure legends and the Material and methods section (Page 17, Line 24 to Page 18 Line 3). EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 8 The data Referee pointed Quantification of data in the revise version Fig. 1C-E (now Fig. 1D) Fig. 1E Fig. 1F Mentioned in the manuscript (Page 5, Line 23-24) Fig. 2D (now Supplementary Fig. 7) Supplementary Fig. 7 Fig. 3H Fig. 3I Fig. 5H (now Fig. 5I) Fig. 5I Fig. 6F Fig. 6G Supplementary Fig. 1 Supplementary Fig. 1 Supplementary Fig. 2 Supplementary Fig. 2 Supplementary Fig. 3 Supplementary Fig. 3 Supplementary Fig. 4 Supplementary Fig. 4 Supplementary Fig. 5 (now Supplementary Fig. 6) Supplementary Fig. 6 Point 3. All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. Response 3: We added the data of cell model experiments without DHT treatment as shown in Table for Referee shown below. The figure in the initial submission The data including DHT-untreated cells in the revised version Fig. 2A Fig. 2C Fig. 2C (now Fig. 2E) Fig. 2F Fig. 2E (now Fig. 2G) Fig. 2I Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig. 6B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Point 4. As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Response 4: In order to explore the role of Hes5 in SBMA, we compared the Hes5 protein and mRNA levels in primary cortical neurons expressing AR24Q and AR97Q using lentivirus infection. AR97Q with DHT reduced the levels of Hes5 both in western blot and RT-qPCR in the primary cortical neurons as shown in NSC97Q (Fig. 8F, G). Furthermore, we confirmed that Hes5 reduction was observed in DHT-treated primary motor neurons expressing AR97Q compared with those expressing AR24Q (Fig. 8H). To further clarify the molecular basis for the beneficial effect of Hes5 in SBMA, we investigated the protein levels of key molecules in SBMA pathogenesis, such as heat shock factor-1 (HSF1), phosphorylated IkBa and Smad2, in NSC34 cells in which Hes5 is depleted. We found that phosphorylation of Smad2 is substantially down-regulated by siRNA-mediated knockdown of Hes5 despite Smad2 protein levels are not altered (Fig. 8A-C). To strengthen the data, we performed additional experiments. As shown in Fig. 8D, pSmad2 was down-regulated in DHT-treated NSC97Q cells compared with DHT-untreated NSC97Q (Fig. 8D). Moreover Hes5 over-expression induced up-regulation of pSmad2 both in NSC97Q and primary cortical neurons (Fig. 8E, I), indicating that Hes5 protects neurons from the toxic insults of polyglutamine-expanded AR via activation of Smad pathway. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 9 Minor points: Point 1. There remain quite a few grammatical errors and typos, the authors should review the manuscript for these Response 1: In response to this comment, our manuscript was proofread by Native speakers of English in Springer Nature Author Services. Point 2. The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? Response 2: In response to the comments, we performed quantitative analysis of Dnmt1 and 5mC in muscle. As shown in Supplementary Fig. 1A, the subcellular localization of Dnmt1 is exclusively in the nucleus. Quantitative analysis confirmed that nuclear levels of Dnmt1 are not altered in AR-97Q mouse (Supplementary Fig. 1B). Furthermore, the level of 5mc was also unaffected in the skeletal muscle of AR-97Q mice (Supplementary Fig. 5A and B). Point 3. Can the authors kindly provide the rationale for choosing RG108? Response 3: A previous study indicated that RG108 was safe and effective in a mouse model of amyotrophic lateral sclerosis (Chestnut BA et al, J Neurosci 2011).This work was also helpful for us to determine the concentration of the agent. Furthermore our preliminary data demonstrated that RG108 improved the viability of SBMA model cells. These findings made us to choose RG108. Point 4. Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. Response 4: Unfortunately, we could not perform blood counts of mice. Therefore, we deleted our description claiming that DNMT inhibitor may be useful in humans (Page 13). Point 5. Figures 3A-D are missing significance labels Response 5: We added the significance labels in Fig. 3A-D. Point 6. Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. Response 6: According to the comment, we explained the backbone and promoter information of Hes5 expression vector and mock vector in the Methods (Page 19, Line 8-9). 2nd Editorial Decision 6 April 2018 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending minor editorial amendments. Please submit your revised manuscript within two weeks. I look forward to seeing a revised form of your manuscript as soon as possible. I look forward to reading a new revised version of your manuscript as soon as possible. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 10 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): The Authors have improved he models used Referee #1 (Remarks for Author): All points were correctly addressed by the Authors Referee #2 (Remarks for Author): The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. 2nd Revision - authors' response 4 May 2018 Referee #1: All points were correctly addressed by the Authors. Response: We are truly grateful to you for your review of our work. Referee #2 The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. Response: We are so grateful to you for the constructive evaluation on our revision. We changed the sentence of conclusions using “possibly” according to the comments (Page 2, Line14). Communication concerning data integrity 17 May 2018 Thank you for sending us the source data for your revised manuscript " DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" at EMBO Molecular Medicine, and for your patience while we evaluated these additional information. We have carefully checked your figures and source data against one another and have come across several inconsistencies that necessitate further investigation. As you may know, the journal classifies image aberrations into three levels (http://embomolmed.embopress.org/classifying-image-aberrations), and we have classified the issues found in your figures collectively as a serious level I/borderline level II. In line with journal policies, this requires that we involve your research institution to provide an opportunity for quality control and investigation at the institutional level. In our experience, an institutional investigation can help greatly in identifying the causes of the apparent aberrations, as institutions can directly review lab books and interview the authors. It is also in your best interest to clarify the issues in a transparent manner with your employer. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 11 We would kindly invite you to let us know who would be the appropriate colleague in charge of research integrity to be contacted at your research institution. I look forward to hearing back form you very soon.

      RESPONSE FROM INSTITUTION AFTER INSTITUTIONAL INVESTIGATION In response to your request, we went through the manuscript by Dr. Masahisa Katsuno and checked the figures at the Ad Hoc Committee of the Research Integrity Committee (RIC) of Nagoya University. As a consequence, we found various mistakes in the figures which should be corrected or reanalyzed. The RIC concluded that the current manuscript does not stand as it is, and recommends the EMBO Molecular Medicine to suspend its publication. 3rd Editorial Decision 12 June 2018 Your manuscript #EMM-2017-08547-V3, entitled "DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" by Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, and Masahisa Katsuno has been withdrawn following recommendations of the Research Integrity Committee of Nagoya University who conducted a formal investigation. Should you be able to fix all issues found by the committee, with formal committee agreement that all issues have been fixed, and as long as that the study remains timely and of interest at the time of submission, we would not be opposed to evaluate it once more for publication as if it was an initial submission. 3rd Revision - authors' response 7 January 2019 CONFIRMATION FROM THE INSTITUTE AFTER AUTHORS PERFORMED REVISION This letter is concerning the manuscript titled ‘DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5’ y Kondo et al (EMM-2017-08547 V4). The research Integrity Committee of Nagoya University confirmed that the authors made revisions in response to comments and concerns by RIC. We agree that the journal reevaluates the appropriateness of the revisions. AUTHOR’S RESPONSE Ad Hoc Committee of the Research Integrity Committee (RIC)

      1. Figure 1. Fig. 1A and the right bottom panel in the source (Dntm3b) Authors’ statements They marked lanes 10, 11, and 12 for Dnmt3b in the original and revised source data. They noticed that the correct lanes were 7, 8, and 9, but have not fixed an error. Conclusion Authors need to fix an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 12 Response 1. We re-performed western blotting for Dnmt3b and confirmed the lanes which we showed in the main figure correspond to the lanes we marked in the revised source file of Fig. 1A. We also revised the quantitative analysis (Fig. 1B) due to the redo of western blot.
      2. Figure 1. Fig. 1G and the right middle panel in the source (GAPDH) Authors’ statements The excised bands for GAPDH in the original figure were wrong. In the revision, they claimed that the dense upper bands were correct GAPDH, and they revised Figure 1G and a frame in the source data accordingly. Concern The predicted molecular weight of GAPDH = 36 kDa. The original bands look correct and the revision looks wrong. Conclusion The authors need to recognize which bands are indeed GAPDH. Response 2. As we mentioned in our response 3 and 4, we re-performed western blotting for Dnmt1 using our original mouse skeletal muscle samples and run the same samples on a separate gel for GAPDH. We confirmed that GAPDH appears just below the molecular weight marker of 37 kDa. We revised Fig. 1G, and marked the right bands in the revised source file of Fig. 1G.
      3. Figure 1. Top two panels in the source for Fig. 1 (Dnmt1) Concern Mobilities of Dnmt1 are different between these two panels. Conclusion Additional analysis is required. The authors need to recognize which bands are indeed Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. Alternatively, the authors may immunoprecipitate Dnmt1 with anti-Dnmt1 antibody #1, and immunoblot the precipitated sample with anti-Dnmt1 antibody #2. Response 3. We noticed that the different results stemmed from the fact that we used antibodies with different Lot numbers. To solve this problem, we tested 3 kinds of antibodies (ab188453, Abcam; ab13537, Abcam; and sc20701, Santa Cruz), and found that ab188453 (1:1000) has the best ability to detect the bands. Using this antibody, we confirmed that Dnmt1 appears between 150 kDa and 250 kDa markers in the western blotting with the sample of NSC97Q cells treated with anti-Dnmt1 siRNA as a negative control (please refer the revised source file for Fig. 1A_Dnmt1). Based on the results of the additional western blotting, we changed the bands of Dnmt1 in Fig. 1A and 1G, and reperformed the quantitative analysis for Fig. 1B and 1H.
      4. Figure 1. Right top and middle panels in the source for Fig. 1 (GAPDH) Concern They first performed Western blotting (WB) with anti-Dnmt1 antibody (right top panel). Without stripping off the anti-Dnmt1 antibody, they reprobed the blot with anti-GAPDH antibody (right middle panel). Dnmt1 WB had many nonspecific bands, and some bands overlapped with GAPDH bands. They cannot estimate the amount of GAPDH without stripping off anti-Dnmt1 antibody. Conclusion Reanalysis is required. Strip off anti-Dnmt1 antibody or run samples on a separate gel for quantification of GAPDH. Response 4. As we mentioned in our Response 2, we re-performed western blotting for Dnmt1 with our original skeletal muscle samples of mice, and run the same samples on another gel for GAPDH to avoid the effects of stripping on re-probing. Based on the results of the additional western blotting, we changed the bands of GAPDH in Fig. 1A and 1G.
      5. Figure 2. Fig. 2A and the left 2nd panel in the source for Fig. 2AC (Dnmt3a) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 13 The molecular weight of Dnmt3a is more than 100 kDa, but looks less than 100 kDa. The RIC assumes that there was an error in the size marker. Conclusion Confirm the size markers and revise the source panel. Response 5. We re-performed western blotting using our original samples and confirmed that the bands of Dnmt3a appear above the marker of 100 kDa. Based on the results of the additional western blotting, we changed the bands of Dnmt3a and GAPDH in Fig. 2A, and re-performed the quantitative analysis.
      6. Figure 2. Fig. 2C and the left 3rd panel in the source for Fig. 2AC (Dnmt1) Authors’ statements Lanes 2-5, 6-9, and 10-13 were from triplicated experiments, and each group of bands looked similar. Dnmt1 in Fig. 2C were excised from lanes 4 and 5, but we inadvertently marked lanes 8 and 9 in the original source data. Conclusion Authors made an appropriate correction in the revised source data. Response 6. To clarify the alteration of Dnmt1 protein levels we re-performed anti-Dnmt1 western blotting using our original samples, and changed the bands of Dnmt1 in Fig. 2C and quantitative data.
      7. Figure 4. Fig. 4C and the left top panel in the source for Fig. 4 (GAPDH) Authors’ statements They could not find the source data for GAPDH in the original Fig. 4C. They thus used GAPDH bands of the same blot with different exposure in the source data. They excised GAPDH bands from the source data, and revised Fig. 4C. The first author looked for the original source data from Toronto, but the limited access to the source data disabled identification of the source data. After he came back to his laboratory, he has found the source data. Conclusion Authors need to make sure that GADPH bands in Fig. 4C and in the revised source data indeed represent correct samples. Response 7. The original data was lost while the first author moved from our lab to Toronto. We thus reperformed anti-AR western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of AR and GAPDH in Fig. 4C.
      8. Figure 4. Fig. 4C and the left bottom panel in the source for Fig. 4 (GAPDH) Concern The mark for 37 kDa does not match the position of a ladder. Conclusion Authors need to confirm the position of size markers. Response 8. As shown in the revised source file of Fig. 4C, we confirmed that GAPDH appears just below the molecular weight maker of 37 kDa.
      9. Figure 4. Fig. 4C and the two left panels in the source for Fig. 4 (Dnmt1 and GAPDH) Authors’ statements Lane marks for Dntm1 and GAPDH were wrong in the original and revised source data. Triplicated experiments were run on lanes 3-4, 5-6, and 7-8. Lanes 3-4 should have been marked, but lanes 2-3 were erroneously marked. Conclusion Authors need to correct an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 14 Response 9. As we mentioned in our response 7,we re-performed anti-AR western blotting, run the same samples on a separate gel for GAPDH (revised Fig. 4C), and re-performed the quantitative analysis (revised Fig. 4D).
      10. Figure 4. Fig. 4C and the top two panels in the source for Fig. 4 (Dnmt1 and Chat) Concern For both panels, triplicated experiments were run on lanes 3-4, 5-6, and 7-8. RG108 appears to similarly increase AR aggregates and Chat in lanes 4 and 8. Similarly, RG108 appears to similarly decrease AR aggregates and Chat lane 6. However, authors conclude that RG108 had no effect on AR aggregation (Fig. 4CD), but significantly increased Chat expression (Fig 4HI). In addition, AR aggregates in Fig. 4C do not appear to identical to the source data in the left upper panel. Conclusion Scrutinized reanalysis is required. The source data do not appear to support the authors' conclusions. Response 10. As explained in our response 7, we re-performed anti-AR western blotting, and confirmed that RG108 did not reduce the high molecular weight complex of AR (revised Fig. 4C). We also reperformed anti-Chat western blotting, and run the same samples for GAPDH. Based on the results of these additional experiments, we changed the bands of Chat and GAPDH (revised Fig. 4H), and reperformed the quantitative analysis (revised Fig. 4I).
      11. Figure 5. Fig. 5A and the right bottom panel in the source for Fig. 5AB (GAPDH) Authors’ statements In the original Fig. 5A, GAPDH bands were erroneously excised from non-specific bands. Correct GAPDH bands were excised in the revision. Conclusion The error was appropriately corrected in the revision. Response 11. To confirm Dnmt1 protein levels using the Dnmt1 antibody (ab188453) we re-performed western blotting using our original samples, and run the same samples for GAPDH to prevent that unstripped bands affect the detection of GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH (revised Fig. 5A), and re-performed the quantitative analysis (revised Fig. 5B).
      12. Figure 7. Fig. 7G and the two right panels in the source for Fig. 7EG (AR monomer and GAPDH) Authors’ statements The source data for AR monomer and GAPDH used in the original Fig. 7G could not be identified in an archive of electronic media. Authors instead found an image with longer exposure time of the same blot. In the revised Fig. 7G, authors excised AR monomer bands and GAPDH bands from the source data with longer exposure time in the revision. Conclusion The author should have archived the source data that they used in their manuscript. However, authors made an acceptable revision. Response 12. None (revision had been made).
      13. Figure 7. Fig. 7EG and the bottom two panels in the source for Fig. 7EG (GAPDH) Concern The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 13. As we mentioned in our response 12, we re-performed anti-GAPDH western blotting, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 15
      14. Figure 7. Figure 7EG and the left top panel in the source for Fig. 7EG (Hes5) Authors’ statements Authors were not aware of the difference between the predicted molecular weight (18 kDa) and the molecular weight of the stained band for Hes5 (~35 kDa). Concern The molecular weight of Hes5 is 18 kDa. The Hes5 bands marked by authors in Figs. 7EG, 8I, and S15BE were ~35 kDa and were much higher than 18 kDa. Datasheets of anti-Hes5 antibody by Novusbio and SantaCruz (M-104) show that Hes5 is stained at ~35 kDa, but the validity of these antibodies need to be scrutinized (Authors used the SantaCruz antibody). In addition, the position of the 50-kDa marker looks incorrect in the source data for Fig. 8I. Conclusion Additional analyses are required. Authors need to recognize the identity of Hes5 by performing Western blotting of cells that are knocked down for Hes5 and/or overexpress Hes5. Knockdown of Hes5 was already shown in S15B, but intensities of multiple bands were reduced. Confirmation by overexpression experiments is likely to be required. Response 14. We clarified that the height of HA-tagged Hes5 is between 20 and 25 kDa in anti-HA western blotting (attached below). We also found that the anti-Hes5 antibody we used in our original experiments (sc13859, Santa Cruz) detects bands between 20 and 25 kDa. Therefore, we re-trimmed such bands in Fig. 8C, 8E, 8F, and 8I, and re-performed the quantitative analysis of them. However, as our original results did not clearly detect Hes5 in Fig7B, 7E, S15B, and S15E, we re-performed additional western blotting of the original samples by shortening the electrophoresis time. According to the Committee’s comments, we run the same samples for western blotting of GAPDH to avoid the effects of stripping. Based on the results of these additional experiments, we changed the bands of Hes5 and GAPDH in Fig. 7B, 7E, S15B, and S15E, and re-performed the quantitative analysis of them. We confirmed that over-expression and knock-down of Hes5 were successfully done in each experiment.
      15. Figure S7. Fig. S7B and the left bottom panel in the source for Fig. S7 (GAPDH) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 16 The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 15. We re-analyzed the original blot, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa (revised source file of Fig S7B).
      16. Figure S7. Fig. S7AC (GAPDH) Concern Source data for GAPDH of Fig. S7AC are not indicated. Conclusion Show the relevant source data. Response 16. We re-performed anti-Dnmt1 western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH in Fig. S7A, and re-performed the quantitative analysis. We also add the result of anti- GAPDH re-probing of the blot shown in Fig. S7C, which was not included in our previous source file.
      17. Figure S7. Fig. S7B, and the right upper and left bottom panels in the source for Fig. S7 (Dnmt3a and GAPDH) Authors’ statements They changed the lane marks on the revised source data, but they claim that the original lane marks were correct. Conclusion Correct the lane marks again. Response 17. We re-marked the right bands in the source file of Fig. S7B.
      18. Figure S7. Fig. S7AC, and the upper two and right bottom panels in the source for Fig. S7 (Dnmt1 and Dnmt3a and Dnmt3b) Authors’ statements The authors marked lanes 5 and 6 in the original source data, but they should have been lanes 3 and
      19. The revised source data were marked correctly. They noticed that the lane marks on the right bottom panel for Dnmt3b should be Fig. S7C, but were erroneously marked as Fig. S7B. Conclusion Correct lanes were marked in the revision. Correct the tag from Fig. S7B to Fig. S7C. Response 18. As we mentioned in our responses 16 and 17, we re-performed western blotting for Fig. S7A, and re-marked the right bands in the source file of Fig. S7B and C, so that the source file corresponds to the Figures. We also corrected the label of Fig. S7C, which had been erroneously shown as “Fig. S7B”.
      20. Figure S7. Fig. S7AB and the left top panel in the source for Fig. S7 (Dnmt1) Concern The authors marked the strongest bands as Dnmt1 in the original and revised source data. However, knockdown of Dnmt1 did not reduce the signal intensity, although the authors showed that the signal intensities were reduced to about a half of controls. Dmnt1 marked in the source data for Figs. 1A, 2A, and 5A was slightly above a 150-kDA marker, whereas Dnmt1 marked in the source data for Fig. 1G was ~250-kDa. In Fig. S7A, the marked Dnmt1 is far above a 150-kDa marker. The RIC can recognize faint bands above the 150-kDa marker and below the strongest bands. Signal intensities of these faint bands were reduced by knockdown of Dnmt1. Conclusion Scrutinized reanalysis is required for Figs. 1A, 1G, 2A, 5A, and S7AB for identity of Dnmt1. This is repetition of concern #3. The authors need to identify Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 17 Response 19. As we mentioned in our responses 3, 6, and 7, we re-performed anti-Dnmt1 western blotting, run the same samples for GAPDH, and re-performed the quantitative analysis for Fig. 1A, 1G, 2C, and S7A. We did so for Fig. 2A, 5A, 5C, and S8A as well.
      21. Figure S15. Figure S15B and the left top panel in the source for Fig. S15 Authors’ statements Molecular weight markers were erroneously marked in all four panels in the original source data. Authors did not scrutinize paring pictures that showed positions of the markers. The markers were corrected in the revision. Conclusion Erroneous marking of molecular weights was appropriately corrected in the revision. Response 20. We re-performed anti-Hes5 western blotting, and confirmed the height of the bands of Hes5 and size markers (revised Fig. S15B and E).
      22. Figure S15. Figure S15BE and top two panels in the source for Fig. S15 (Hes5) Authors’ statements Bands for Hes5 (S15BE) and Gapdh (S15B) were erroneously marked in the original source data, and were corrected in the revision. Concern The concern is identical to concern #13. The indicated molecular weight of Hes5 (~35 kDa) is much different from it predicted molecular weight of 18 kDa. Conclusion Additional analyses are required. Erroneous marking of bands for Hes5 and Gapdh were corrected in the revision, but identity of Hes5 should be revealed. Response 21. As we mentioned in our response 20, we re-performed anti-Hes5 western blotting, and confirmed that the height of the bands of Hes5 is between 20 and 25 kDa (revised source file of Fig. S15B and E).
      23. Results for Figure 6FG (Hes5 expression after RG108 treatment) Concern In a section for “RG108 recovers Hes5 expression” in Results, authors state “it (Hes5) was diminished by RG108 treatment (Fig. 6F and G)”. This should be “enhanced” or “upregulated”. Conclusion The error should be corrected. Response 22. We corrected “diminished” to “up-regulated” in the text (Page 10, Line 13). 4th Editorial Decision 11 January 2019 Thank you for submitting your newly revised manuscript to EMBO Molecular Medicine. We have now carefully evaluated the figures along with the source data provided as well as the reply to the RIC letter that you kindly provided. While in principle, we are supportive of publication, still a few items remain to be corrected and some have to be added as follows: 1) Figures vs. source data: please fix!
      24. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 18 Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q
      25. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples I look forward to receiving a new revised version of your manuscript as soon as possible. 4th Revision - authors' response 1 March 2019 We are most grateful to you for your supportive comments on our manuscript. We revised Figures, Source data and manuscript according to the comment 1) 2) 5) and 6), and added created Author checklist, The Paper Explained, Synopsis, and Visual abstract in response to the comments 3) 4) 7) 8) and 9). Detailed responses are shown below. 1) Figures vs. source data: please fix!
      26. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q Response: In accordance to your instruction, we revised the Figures so that they match the source data, by removing any manipulation of angle and contrast of the images.
      27. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples Response: As you pointed out we had a mistake to label the data. We changed the label in the source of Appendix Figure S3B_GAPDH from “cerebellum” to “cortex”. USEFUL LINKS FOR COMPLETING THIS FORM http://www.antibodypedia.com http://1degreebio.org http://www.equator-network.org/reporting-guidelines/improving-bioscience-research-reporting-the-arrive-guidelines-for-reporting-http://grants.nih.gov/grants/olaw/olaw.htm http://www.mrc.ac.uk/Ourresearch/Ethicsresearchguidance/Useofanimals/index.htm http://ClinicalTrials.gov http://www.consort-statement.org http://www.consort-statement.org/checklists/view/32-consort/66-title ! http://www.equator-network.org/reporting-guidelines/reporting-recommendations-for-tumour-marker-prognostic-studies-! http://datadryad.org ! http://figshare.com ! http://www.ncbi.nlm.nih.gov/gap ! http://www.ebi.ac.uk/ega http://biomodels.net/ http://biomodels.net/miriam/ ! http://jjj.biochem.sun.ac.za ! http://oba.od.nih.gov/biosecurity/biosecurity_documents.html ! http://www.selectagents.gov/ ! ! ! ! ! " common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; " are tests one-sided or two-sided? " are there adjustments for multiple comparisons? " exact statistical test results, e.g., P values = x but not P values < x; " definition of ‘center values’ as median or average; " definition of error bars as s.d. or s.e.m. 1.a. How was the sample size chosen to ensure adequate power to detect a pre-specified effect size? 1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
      28. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria preestablished?
      29. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe. For animal studies, include a statement about randomization even if no randomization was used. 4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. 4.b. For animal studies, include a statement about blinding even if no blinding was done
      30. For every figure, are statistical tests justified as appropriate? Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Is there an estimate of variation within each group of data? Is the variance similar between the groups that are being statistically compared? Manuscript Number: EMM-2017-08547 EMBO PRESS A- Figures Reporting Checklist For Life Sciences Articles (Rev. June 2017) This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal’s authorship guidelines in preparing your manuscript. PLEASE NOTE THAT THIS CHECKLIST WILL BE PUBLISHED ALONGSIDE YOUR PAPER Journal Submitted to: EMBO molecular medicine Corresponding Author Name: Masahisa Katsuno a statement of how many times the experiment shown was independently replicated in the laboratory. Any descriptions too long for the figure legend should be included in the methods section and/or with the source data. In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects. definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.). Please fill out these boxes # (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name). C- Reagents B- Statistics and general methods the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
      31. Data the data were obtained and processed according to the field’s best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:
      32. Captions The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation. YOU MUST COMPLETE ALL CELLS WITH A PINK BACKGROUND # Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). NA We randomly allocated the mice to each treatment group (Page 15). We randomly allocated the mice to each treatment group (Page 15). The investigater blindly performed the behavior tests (Page 15). The investigater blindly performed the behavior tests (Page 15). Yes. We described the statistical methods in each legend. Yes. We confirmed the normality of data. Yes. We tested them using ANOVA. Yes. We confirmed the variance was similar between groups.
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      36. Report species, strain, gender, age of animals and genetic modification status where applicable. Please detail housing and husbandry conditions and the source of animals.
      37. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the committee(s) approving the experiments.
      38. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, 2010) to ensure that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.
      39. Identify the committee(s) approving the study protocol.
      40. Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
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      45. For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right). See author guidelines, under ‘Reporting Guidelines’. Please confirm you have followed these guidelines. 18: Provide a “Data Availability” section at the end of the Materials & Methods, listing the accession codes for data generated in this study and deposited in a public database (e.g. RNA-Seq data: Gene Expression Omnibus GSE39462, Proteomics data: PRIDE PXD000208 etc.) Please refer to our author guidelines for ‘Data Deposition’. Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Functional genomics data e. Proteomics and molecular interactions
      46. Deposition is strongly recommended for any datasets that are central and integral to the study; please consider the journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right).
      47. Access to human clinical and genomic datasets should be provided with as few restrictions as possible while respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public accesscontrolled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right).
      48. Computational models that are central and integral to a study should be shared without restrictions and provided in a machine-readable form. The relevant accession numbers or links should be provided. When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.
      49. Could your study fall under dual use research restrictions? Please check biosecurity documents (see link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could. F- Data Accessibility D- Animal Models E- Human Subjects We confirmed the guideline. G- Dual use research of concern We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page16-17 (section of Immunoblotting, and Histology and immunohistochemistry in the Material and Method). We described the details of cell lines on Page 6,8,9,and 22 (section of Result and Material and Method). We tested mycoplasma contamination. We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). NA NA NA NA NA NA NA NA
    2. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 1 DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5 Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, Masahisa Katsuno Review timeline: Submission date: 1 October 2017 Editorial Decision: 13 November 2017 Revision received: 27 February 2018 Editorial Decision: 6 April 2018 Revision received: 4 May 2018 Extra communication: 17 May 2018 Editorial Decision: 12 June 2018 Revision received: 7 January 2019 Editorial Decision: 11 Jannuary 2019 Revision received: 1 March 2019 Accepted: 1 March 2019 Editor: Céline Carret Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 13 November 2017 Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the two referees whom we asked to evaluate your manuscript. As you will see from the reports below, the referees find the data intriguing. Unfortunately, at this stage the work needs to be considerably strengthen to be more conclusive, and both referees provide good suggestions for that. Additional controls and experiments and adding mechanistic understanding seem to be required for the paper to be further evaluated in EMBO Molecular Medicine. Given that the referees find the message interesting, we would be willing to consider a revised manuscript with the understanding that the referee concerns must be fully addressed and that acceptance of the manuscript would entail a second round of review. I should remind you that EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. I realize that addressing the referees' comments in full would involve a lot of additional experimental work and resources and I am uncertain whether you will be able (or willing) to return a revised manuscript within the 3 months deadline. I would also understand your decision if you chose to rather seek rapid publication elsewhere at this stage. I look forward to seeing a revised form of your manuscript as soon as possible. Please read below for important editorial formatting and consult our author's guidelines for proper formatting of your revised article for EMBO Molecular Medicine. Should you find that the requested revisions are not feasible within the constraints outlined here and choose, therefore, to submit your paper elsewhere, we would welcome a message to this effect. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 2 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): Better control should be used as detailed to Authors Referee #1 (Remarks for Author): The study by Kondo al coll. Reports that the toxicity of the mutant AR responsible for SBMA could be ascribed to altered DNA methylation, and that this alteration could be reverted by the inhibition of this mechanisms using DNA methylation inhibitor such as RG108. In addition, the Authors have demonstrated that the hyper-methylation of the Hes5 gene correlates with the AR-97Q toxicity and that overexpression of Hes5 rescues from SBMA phenotype in cell models of the disease. While the study is potentially interesting, several points must be addressed in order to definitely prove that DNA methylation is relevant in SBMA. Major points:

      1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice.
      2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses.
      3. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motorneurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT.
      4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript
      5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells?
      6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108 EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 3
      7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers.
      8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice?
      9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR- 24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Minor Points
      10. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR.
      11. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Referee #2 (Comments on Novelty/Model System for Author): See comments to authors for 1 and 4. One issue is that validation in the NSC transformed cell line model is not sufficient. Referee #2 (Remarks for Author): In the Kondo et al. paper, a role for altered methylation in the polyglutamine disease SBMA is sought. The authors show Dnmt1 is upregulated within spinal motor neurons and suppression by siRNA or by a chemical inhibitor RG108 can suppress neuronal cell death. The authors translated the inhibitor into a pre-clinical mouse model and found evidence for suppression of SBMA neurodegenerative phenotypes. Intriguingly, this occurred downstream of polyglutamine aggregation, as aggregation was not affected. The authors show that cell viability in a NSC cell model of SBMA enabled by RG108 treatment depends on Hes5 expression, and that re-expression of Hes5 is sufficient to rescue viability. While the topic of methylation in neurodegeneration has not been addressed much, making the work intriguing, there are a number of problems with the investigation that undermine one's confidence in the results. The authors' explanation for which genes are affected by altered methylation is very superficial, and what Hes5 is doing is entirely unclear. As the validation of Hes5 takes place in only the transformed cell line model of SBMA, it seems rather premature to implicate Hes5 without providing a more thorough understanding of what it is doing. More concrete data is needed to balance what appears at times as speculation. Specific concerns that should be addressed to strengthen the manuscript are as follows: 1) The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. 2) A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and reviewers) can determine if the conclusions are valid. The problematic Figures are as follows:
      12. Fig 1C
      13. Fig 1D
      14. Fig 1E, EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 4
      15. Fig 1F
      16. Fig 2D
      17. Fig 3H
      18. Fig 5H
      19. Fig 6F
      20. Fig S1
      21. Fig S2
      22. Fig S3
      23. Fig S4
      24. Fig S5 3) All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. 4) As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Minor points: -There remain quite a few grammatical errors and typos, the authors should review the manuscript for these -The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? -Can the authors kindly provide the rationale for choosing RG108? -Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. -Figures 3A-D are missing significance labels -Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. 1st Revision - authors' response 27 February 2018 Referee #1: Major points: Point 1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice. Response 1: We thank the Referee for this important suggestion. In response to the proposal, we performed RT-qPCR of Dnmt1, Dnmt3a, and Dnmt3b using spinal cord samples from each group of mice (Fig. 1C). This analysis revealed that Dnmt1 mRNA level was up-regulated in AR-97Q mice as well as protein level. Point 2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 5 Response 2: In response to the Referee’s comment, we added the data of AR-24Q mice in Fig. 1A, 1B, 1C, 1D, 1E, 5I, and 5J, so that we excluded the influence of normal AR to the protein level and mRNA level of Dnmt1, Dnmt3a, Dnmt3b, and Hes5. Point 3-1. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. Response 3-1: We changed the term “expression” to “protein level” or “level” for the part of manuscript describing western blotting. In order to evaluate the gene expression level of Dnmt1, we performed RT-qPCR of NSC24Q and NSC97Q cells, and confirmed that Dnmt1 mRNA is expressed at a higher level in NSC97Q compared with NSC24Q (Fig. 2B). Point 3-2. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motor neurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. Response 3-2: We changed the sentence according to Referee’s suggestion (Page 7, Line 1-2). Point 3-3. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT. Response 3-3: To address the comment, we added the data comparing protein levels of Dnmts and Dnmt1 mRNA level in NSC97Q with or without DHT treatment (Fig. 2C and D). Moreover, we analyzed the influence of Dnmts knockdown on the cell viability of DHT-untreatd NSC97Q cells (Fig. 2F). Furthermore we evaluate the effect of RG108 to NSC97Q cells without DHT (Fig. 2I). The results of these additional experiments showed that Dnmt1 is not up-regulated in DHTuntreated NSC97Q cells, and that neither siRNA-mediated knockdown of Dnmts nor RG108 treatment alters the cell viability of NSC97Q cells without DHT treatment. Point 4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript. Response 4: According the Referee’s suggestion, we added the data of WST-8 assay of DHTtreated NSC24Q cells with siRNA-mediated knockdown of Dnmts (Supplementary Fig. 9) and RG108 treatment to DHT-treated NSC24Q (Fig. 2H and J). Both knockdown of Dnmts and RG108 had no effect on the cell viability of NSC24Q with DHT treatment. Point 5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells? Response 5: The DNA methylation array analysis we utilized was developed for human materials, but could not be applied to mouse samples at the time we performed experiments. Therefore we analyzed the samples of SH-SY5Y, human neuronal cells. In order to confirm that the phenomena observed in human cells are reproduced in murine cells, we performed all the experiments using cell samples derived from SH-SY5Y (Fig. 5 A, 5B, 5C, 5D, 5F, 5G, and 5H; Supplementary Fig. 12; Fig. 6A and 6B) and those from NSC34 (Fig. 2A and 2C; Supplementary Fig.13 and Fig. 6 C and 6D) in parallel. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 6 Point 6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108. Response 6: Our preliminary experiments revealed that Dnmt1 protein level was already elevated in the spinal cord of 6-week-old AR-97Q mouse (Figure for Referee shown below). This is the reason why we decided to start the brain injection treatment with RG108 to 6-week-old age mice. Point 7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers. Response 7: We changed the sentence to “DNA methylation array analysis using these cells revealed that DNA methylation of CpG islands is intensified in several genes. However, total DNA methylation level was not altered between SH24Q and SH97Q (Supplementary Fig. 11).” (Page 9, Line 7-9) Point 8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice? Response 8: To address this issue, we performed western blot using nuclear and cytoplasmic fraction of spinal cord lysate obtained from wild-type and AR97Q mice. Both in wild-type and AR97Q mice, Hes5 showed cytoplasm-dominant localization (Figure for Referee shown below). Although we cannot exclude the possibility that nuclear localization of Hes5 is somehow impaired in AR97Q mice, the western blot indicates that Hes5 is down-regulated both in nucleus and cytoplasm in SBMA model mouse, presumably resulting in the faint nuclear staining in the immunohistochemistry of the AR97Q mice. Point 9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR-24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Response 9: According to the comment, we added the data comparing DHT(-) and DHT(+) as shown in the Table for Referee shown below. As AR aggregation is not detectable in NSC97Q cell without DHT treatment, we did not perform the counterpart experiment for Fig. 7G and H. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 7 The data Referee pointed The corresponding data in the revise version Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig6. B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Minor Points Point 1. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR. Response: We are thankful to the suggestion. We changed RT-PCR to RT-qPCR throughout the manuscript. Point 2. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Response: We changed the term from “Wild” to “Wild-type”. Referee #2 Point 1. The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. Response 1: We thank the Referee for pointing this important issue. We now confirmed that relative mRNA level of human AR was not altered by RG108 treatment (Fig. 4E). This data indicated that RG108 had does not suppress AR transgene expression. Point 2. A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and Referees) can determine if the conclusions are valid. The problematic Figures are as follows:
      25. Fig 1C
      26. Fig 1D
      27. Fig 1E,
      28. Fig 1F
      29. Fig 2D
      30. Fig 3H
      31. Fig 5H
      32. Fig 6F
      33. Fig S1
      34. Fig S2
      35. Fig S3
      36. Fig S4
      37. Fig S5 Response 2: In response to the comment, we quantified the data as described below. Furthermore, we described the number of samples analyzed for quantitation in the figure legends and the Material and methods section (Page 17, Line 24 to Page 18 Line 3). EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 8 The data Referee pointed Quantification of data in the revise version Fig. 1C-E (now Fig. 1D) Fig. 1E Fig. 1F Mentioned in the manuscript (Page 5, Line 23-24) Fig. 2D (now Supplementary Fig. 7) Supplementary Fig. 7 Fig. 3H Fig. 3I Fig. 5H (now Fig. 5I) Fig. 5I Fig. 6F Fig. 6G Supplementary Fig. 1 Supplementary Fig. 1 Supplementary Fig. 2 Supplementary Fig. 2 Supplementary Fig. 3 Supplementary Fig. 3 Supplementary Fig. 4 Supplementary Fig. 4 Supplementary Fig. 5 (now Supplementary Fig. 6) Supplementary Fig. 6 Point 3. All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. Response 3: We added the data of cell model experiments without DHT treatment as shown in Table for Referee shown below. The figure in the initial submission The data including DHT-untreated cells in the revised version Fig. 2A Fig. 2C Fig. 2C (now Fig. 2E) Fig. 2F Fig. 2E (now Fig. 2G) Fig. 2I Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig. 6B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Point 4. As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Response 4: In order to explore the role of Hes5 in SBMA, we compared the Hes5 protein and mRNA levels in primary cortical neurons expressing AR24Q and AR97Q using lentivirus infection. AR97Q with DHT reduced the levels of Hes5 both in western blot and RT-qPCR in the primary cortical neurons as shown in NSC97Q (Fig. 8F, G). Furthermore, we confirmed that Hes5 reduction was observed in DHT-treated primary motor neurons expressing AR97Q compared with those expressing AR24Q (Fig. 8H). To further clarify the molecular basis for the beneficial effect of Hes5 in SBMA, we investigated the protein levels of key molecules in SBMA pathogenesis, such as heat shock factor-1 (HSF1), phosphorylated IkBa and Smad2, in NSC34 cells in which Hes5 is depleted. We found that phosphorylation of Smad2 is substantially down-regulated by siRNA-mediated knockdown of Hes5 despite Smad2 protein levels are not altered (Fig. 8A-C). To strengthen the data, we performed additional experiments. As shown in Fig. 8D, pSmad2 was down-regulated in DHT-treated NSC97Q cells compared with DHT-untreated NSC97Q (Fig. 8D). Moreover Hes5 over-expression induced up-regulation of pSmad2 both in NSC97Q and primary cortical neurons (Fig. 8E, I), indicating that Hes5 protects neurons from the toxic insults of polyglutamine-expanded AR via activation of Smad pathway. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 9 Minor points: Point 1. There remain quite a few grammatical errors and typos, the authors should review the manuscript for these Response 1: In response to this comment, our manuscript was proofread by Native speakers of English in Springer Nature Author Services. Point 2. The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? Response 2: In response to the comments, we performed quantitative analysis of Dnmt1 and 5mC in muscle. As shown in Supplementary Fig. 1A, the subcellular localization of Dnmt1 is exclusively in the nucleus. Quantitative analysis confirmed that nuclear levels of Dnmt1 are not altered in AR-97Q mouse (Supplementary Fig. 1B). Furthermore, the level of 5mc was also unaffected in the skeletal muscle of AR-97Q mice (Supplementary Fig. 5A and B). Point 3. Can the authors kindly provide the rationale for choosing RG108? Response 3: A previous study indicated that RG108 was safe and effective in a mouse model of amyotrophic lateral sclerosis (Chestnut BA et al, J Neurosci 2011).This work was also helpful for us to determine the concentration of the agent. Furthermore our preliminary data demonstrated that RG108 improved the viability of SBMA model cells. These findings made us to choose RG108. Point 4. Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. Response 4: Unfortunately, we could not perform blood counts of mice. Therefore, we deleted our description claiming that DNMT inhibitor may be useful in humans (Page 13). Point 5. Figures 3A-D are missing significance labels Response 5: We added the significance labels in Fig. 3A-D. Point 6. Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. Response 6: According to the comment, we explained the backbone and promoter information of Hes5 expression vector and mock vector in the Methods (Page 19, Line 8-9). 2nd Editorial Decision 6 April 2018 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending minor editorial amendments. Please submit your revised manuscript within two weeks. I look forward to seeing a revised form of your manuscript as soon as possible. I look forward to reading a new revised version of your manuscript as soon as possible. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 10 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): The Authors have improved he models used Referee #1 (Remarks for Author): All points were correctly addressed by the Authors Referee #2 (Remarks for Author): The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. 2nd Revision - authors' response 4 May 2018 Referee #1: All points were correctly addressed by the Authors. Response: We are truly grateful to you for your review of our work. Referee #2 The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. Response: We are so grateful to you for the constructive evaluation on our revision. We changed the sentence of conclusions using “possibly” according to the comments (Page 2, Line14). Communication concerning data integrity 17 May 2018 Thank you for sending us the source data for your revised manuscript " DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" at EMBO Molecular Medicine, and for your patience while we evaluated these additional information. We have carefully checked your figures and source data against one another and have come across several inconsistencies that necessitate further investigation. As you may know, the journal classifies image aberrations into three levels (http://embomolmed.embopress.org/classifying-image-aberrations), and we have classified the issues found in your figures collectively as a serious level I/borderline level II. In line with journal policies, this requires that we involve your research institution to provide an opportunity for quality control and investigation at the institutional level. In our experience, an institutional investigation can help greatly in identifying the causes of the apparent aberrations, as institutions can directly review lab books and interview the authors. It is also in your best interest to clarify the issues in a transparent manner with your employer. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 11 We would kindly invite you to let us know who would be the appropriate colleague in charge of research integrity to be contacted at your research institution. I look forward to hearing back form you very soon.

      RESPONSE FROM INSTITUTION AFTER INSTITUTIONAL INVESTIGATION In response to your request, we went through the manuscript by Dr. Masahisa Katsuno and checked the figures at the Ad Hoc Committee of the Research Integrity Committee (RIC) of Nagoya University. As a consequence, we found various mistakes in the figures which should be corrected or reanalyzed. The RIC concluded that the current manuscript does not stand as it is, and recommends the EMBO Molecular Medicine to suspend its publication. 3rd Editorial Decision 12 June 2018 Your manuscript #EMM-2017-08547-V3, entitled "DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" by Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, and Masahisa Katsuno has been withdrawn following recommendations of the Research Integrity Committee of Nagoya University who conducted a formal investigation. Should you be able to fix all issues found by the committee, with formal committee agreement that all issues have been fixed, and as long as that the study remains timely and of interest at the time of submission, we would not be opposed to evaluate it once more for publication as if it was an initial submission. 3rd Revision - authors' response 7 January 2019 CONFIRMATION FROM THE INSTITUTE AFTER AUTHORS PERFORMED REVISION This letter is concerning the manuscript titled ‘DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5’ y Kondo et al (EMM-2017-08547 V4). The research Integrity Committee of Nagoya University confirmed that the authors made revisions in response to comments and concerns by RIC. We agree that the journal reevaluates the appropriateness of the revisions. AUTHOR’S RESPONSE Ad Hoc Committee of the Research Integrity Committee (RIC)

      1. Figure 1. Fig. 1A and the right bottom panel in the source (Dntm3b) Authors’ statements They marked lanes 10, 11, and 12 for Dnmt3b in the original and revised source data. They noticed that the correct lanes were 7, 8, and 9, but have not fixed an error. Conclusion Authors need to fix an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 12 Response 1. We re-performed western blotting for Dnmt3b and confirmed the lanes which we showed in the main figure correspond to the lanes we marked in the revised source file of Fig. 1A. We also revised the quantitative analysis (Fig. 1B) due to the redo of western blot.
      2. Figure 1. Fig. 1G and the right middle panel in the source (GAPDH) Authors’ statements The excised bands for GAPDH in the original figure were wrong. In the revision, they claimed that the dense upper bands were correct GAPDH, and they revised Figure 1G and a frame in the source data accordingly. Concern The predicted molecular weight of GAPDH = 36 kDa. The original bands look correct and the revision looks wrong. Conclusion The authors need to recognize which bands are indeed GAPDH. Response 2. As we mentioned in our response 3 and 4, we re-performed western blotting for Dnmt1 using our original mouse skeletal muscle samples and run the same samples on a separate gel for GAPDH. We confirmed that GAPDH appears just below the molecular weight marker of 37 kDa. We revised Fig. 1G, and marked the right bands in the revised source file of Fig. 1G.
      3. Figure 1. Top two panels in the source for Fig. 1 (Dnmt1) Concern Mobilities of Dnmt1 are different between these two panels. Conclusion Additional analysis is required. The authors need to recognize which bands are indeed Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. Alternatively, the authors may immunoprecipitate Dnmt1 with anti-Dnmt1 antibody #1, and immunoblot the precipitated sample with anti-Dnmt1 antibody #2. Response 3. We noticed that the different results stemmed from the fact that we used antibodies with different Lot numbers. To solve this problem, we tested 3 kinds of antibodies (ab188453, Abcam; ab13537, Abcam; and sc20701, Santa Cruz), and found that ab188453 (1:1000) has the best ability to detect the bands. Using this antibody, we confirmed that Dnmt1 appears between 150 kDa and 250 kDa markers in the western blotting with the sample of NSC97Q cells treated with anti-Dnmt1 siRNA as a negative control (please refer the revised source file for Fig. 1A_Dnmt1). Based on the results of the additional western blotting, we changed the bands of Dnmt1 in Fig. 1A and 1G, and reperformed the quantitative analysis for Fig. 1B and 1H.
      4. Figure 1. Right top and middle panels in the source for Fig. 1 (GAPDH) Concern They first performed Western blotting (WB) with anti-Dnmt1 antibody (right top panel). Without stripping off the anti-Dnmt1 antibody, they reprobed the blot with anti-GAPDH antibody (right middle panel). Dnmt1 WB had many nonspecific bands, and some bands overlapped with GAPDH bands. They cannot estimate the amount of GAPDH without stripping off anti-Dnmt1 antibody. Conclusion Reanalysis is required. Strip off anti-Dnmt1 antibody or run samples on a separate gel for quantification of GAPDH. Response 4. As we mentioned in our Response 2, we re-performed western blotting for Dnmt1 with our original skeletal muscle samples of mice, and run the same samples on another gel for GAPDH to avoid the effects of stripping on re-probing. Based on the results of the additional western blotting, we changed the bands of GAPDH in Fig. 1A and 1G.
      5. Figure 2. Fig. 2A and the left 2nd panel in the source for Fig. 2AC (Dnmt3a) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 13 The molecular weight of Dnmt3a is more than 100 kDa, but looks less than 100 kDa. The RIC assumes that there was an error in the size marker. Conclusion Confirm the size markers and revise the source panel. Response 5. We re-performed western blotting using our original samples and confirmed that the bands of Dnmt3a appear above the marker of 100 kDa. Based on the results of the additional western blotting, we changed the bands of Dnmt3a and GAPDH in Fig. 2A, and re-performed the quantitative analysis.
      6. Figure 2. Fig. 2C and the left 3rd panel in the source for Fig. 2AC (Dnmt1) Authors’ statements Lanes 2-5, 6-9, and 10-13 were from triplicated experiments, and each group of bands looked similar. Dnmt1 in Fig. 2C were excised from lanes 4 and 5, but we inadvertently marked lanes 8 and 9 in the original source data. Conclusion Authors made an appropriate correction in the revised source data. Response 6. To clarify the alteration of Dnmt1 protein levels we re-performed anti-Dnmt1 western blotting using our original samples, and changed the bands of Dnmt1 in Fig. 2C and quantitative data.
      7. Figure 4. Fig. 4C and the left top panel in the source for Fig. 4 (GAPDH) Authors’ statements They could not find the source data for GAPDH in the original Fig. 4C. They thus used GAPDH bands of the same blot with different exposure in the source data. They excised GAPDH bands from the source data, and revised Fig. 4C. The first author looked for the original source data from Toronto, but the limited access to the source data disabled identification of the source data. After he came back to his laboratory, he has found the source data. Conclusion Authors need to make sure that GADPH bands in Fig. 4C and in the revised source data indeed represent correct samples. Response 7. The original data was lost while the first author moved from our lab to Toronto. We thus reperformed anti-AR western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of AR and GAPDH in Fig. 4C.
      8. Figure 4. Fig. 4C and the left bottom panel in the source for Fig. 4 (GAPDH) Concern The mark for 37 kDa does not match the position of a ladder. Conclusion Authors need to confirm the position of size markers. Response 8. As shown in the revised source file of Fig. 4C, we confirmed that GAPDH appears just below the molecular weight maker of 37 kDa.
      9. Figure 4. Fig. 4C and the two left panels in the source for Fig. 4 (Dnmt1 and GAPDH) Authors’ statements Lane marks for Dntm1 and GAPDH were wrong in the original and revised source data. Triplicated experiments were run on lanes 3-4, 5-6, and 7-8. Lanes 3-4 should have been marked, but lanes 2-3 were erroneously marked. Conclusion Authors need to correct an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 14 Response 9. As we mentioned in our response 7,we re-performed anti-AR western blotting, run the same samples on a separate gel for GAPDH (revised Fig. 4C), and re-performed the quantitative analysis (revised Fig. 4D).
      10. Figure 4. Fig. 4C and the top two panels in the source for Fig. 4 (Dnmt1 and Chat) Concern For both panels, triplicated experiments were run on lanes 3-4, 5-6, and 7-8. RG108 appears to similarly increase AR aggregates and Chat in lanes 4 and 8. Similarly, RG108 appears to similarly decrease AR aggregates and Chat lane 6. However, authors conclude that RG108 had no effect on AR aggregation (Fig. 4CD), but significantly increased Chat expression (Fig 4HI). In addition, AR aggregates in Fig. 4C do not appear to identical to the source data in the left upper panel. Conclusion Scrutinized reanalysis is required. The source data do not appear to support the authors' conclusions. Response 10. As explained in our response 7, we re-performed anti-AR western blotting, and confirmed that RG108 did not reduce the high molecular weight complex of AR (revised Fig. 4C). We also reperformed anti-Chat western blotting, and run the same samples for GAPDH. Based on the results of these additional experiments, we changed the bands of Chat and GAPDH (revised Fig. 4H), and reperformed the quantitative analysis (revised Fig. 4I).
      11. Figure 5. Fig. 5A and the right bottom panel in the source for Fig. 5AB (GAPDH) Authors’ statements In the original Fig. 5A, GAPDH bands were erroneously excised from non-specific bands. Correct GAPDH bands were excised in the revision. Conclusion The error was appropriately corrected in the revision. Response 11. To confirm Dnmt1 protein levels using the Dnmt1 antibody (ab188453) we re-performed western blotting using our original samples, and run the same samples for GAPDH to prevent that unstripped bands affect the detection of GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH (revised Fig. 5A), and re-performed the quantitative analysis (revised Fig. 5B).
      12. Figure 7. Fig. 7G and the two right panels in the source for Fig. 7EG (AR monomer and GAPDH) Authors’ statements The source data for AR monomer and GAPDH used in the original Fig. 7G could not be identified in an archive of electronic media. Authors instead found an image with longer exposure time of the same blot. In the revised Fig. 7G, authors excised AR monomer bands and GAPDH bands from the source data with longer exposure time in the revision. Conclusion The author should have archived the source data that they used in their manuscript. However, authors made an acceptable revision. Response 12. None (revision had been made).
      13. Figure 7. Fig. 7EG and the bottom two panels in the source for Fig. 7EG (GAPDH) Concern The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 13. As we mentioned in our response 12, we re-performed anti-GAPDH western blotting, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 15
      14. Figure 7. Figure 7EG and the left top panel in the source for Fig. 7EG (Hes5) Authors’ statements Authors were not aware of the difference between the predicted molecular weight (18 kDa) and the molecular weight of the stained band for Hes5 (~35 kDa). Concern The molecular weight of Hes5 is 18 kDa. The Hes5 bands marked by authors in Figs. 7EG, 8I, and S15BE were ~35 kDa and were much higher than 18 kDa. Datasheets of anti-Hes5 antibody by Novusbio and SantaCruz (M-104) show that Hes5 is stained at ~35 kDa, but the validity of these antibodies need to be scrutinized (Authors used the SantaCruz antibody). In addition, the position of the 50-kDa marker looks incorrect in the source data for Fig. 8I. Conclusion Additional analyses are required. Authors need to recognize the identity of Hes5 by performing Western blotting of cells that are knocked down for Hes5 and/or overexpress Hes5. Knockdown of Hes5 was already shown in S15B, but intensities of multiple bands were reduced. Confirmation by overexpression experiments is likely to be required. Response 14. We clarified that the height of HA-tagged Hes5 is between 20 and 25 kDa in anti-HA western blotting (attached below). We also found that the anti-Hes5 antibody we used in our original experiments (sc13859, Santa Cruz) detects bands between 20 and 25 kDa. Therefore, we re-trimmed such bands in Fig. 8C, 8E, 8F, and 8I, and re-performed the quantitative analysis of them. However, as our original results did not clearly detect Hes5 in Fig7B, 7E, S15B, and S15E, we re-performed additional western blotting of the original samples by shortening the electrophoresis time. According to the Committee’s comments, we run the same samples for western blotting of GAPDH to avoid the effects of stripping. Based on the results of these additional experiments, we changed the bands of Hes5 and GAPDH in Fig. 7B, 7E, S15B, and S15E, and re-performed the quantitative analysis of them. We confirmed that over-expression and knock-down of Hes5 were successfully done in each experiment.
      15. Figure S7. Fig. S7B and the left bottom panel in the source for Fig. S7 (GAPDH) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 16 The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 15. We re-analyzed the original blot, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa (revised source file of Fig S7B).
      16. Figure S7. Fig. S7AC (GAPDH) Concern Source data for GAPDH of Fig. S7AC are not indicated. Conclusion Show the relevant source data. Response 16. We re-performed anti-Dnmt1 western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH in Fig. S7A, and re-performed the quantitative analysis. We also add the result of anti- GAPDH re-probing of the blot shown in Fig. S7C, which was not included in our previous source file.
      17. Figure S7. Fig. S7B, and the right upper and left bottom panels in the source for Fig. S7 (Dnmt3a and GAPDH) Authors’ statements They changed the lane marks on the revised source data, but they claim that the original lane marks were correct. Conclusion Correct the lane marks again. Response 17. We re-marked the right bands in the source file of Fig. S7B.
      18. Figure S7. Fig. S7AC, and the upper two and right bottom panels in the source for Fig. S7 (Dnmt1 and Dnmt3a and Dnmt3b) Authors’ statements The authors marked lanes 5 and 6 in the original source data, but they should have been lanes 3 and
      19. The revised source data were marked correctly. They noticed that the lane marks on the right bottom panel for Dnmt3b should be Fig. S7C, but were erroneously marked as Fig. S7B. Conclusion Correct lanes were marked in the revision. Correct the tag from Fig. S7B to Fig. S7C. Response 18. As we mentioned in our responses 16 and 17, we re-performed western blotting for Fig. S7A, and re-marked the right bands in the source file of Fig. S7B and C, so that the source file corresponds to the Figures. We also corrected the label of Fig. S7C, which had been erroneously shown as “Fig. S7B”.
      20. Figure S7. Fig. S7AB and the left top panel in the source for Fig. S7 (Dnmt1) Concern The authors marked the strongest bands as Dnmt1 in the original and revised source data. However, knockdown of Dnmt1 did not reduce the signal intensity, although the authors showed that the signal intensities were reduced to about a half of controls. Dmnt1 marked in the source data for Figs. 1A, 2A, and 5A was slightly above a 150-kDA marker, whereas Dnmt1 marked in the source data for Fig. 1G was ~250-kDa. In Fig. S7A, the marked Dnmt1 is far above a 150-kDa marker. The RIC can recognize faint bands above the 150-kDa marker and below the strongest bands. Signal intensities of these faint bands were reduced by knockdown of Dnmt1. Conclusion Scrutinized reanalysis is required for Figs. 1A, 1G, 2A, 5A, and S7AB for identity of Dnmt1. This is repetition of concern #3. The authors need to identify Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 17 Response 19. As we mentioned in our responses 3, 6, and 7, we re-performed anti-Dnmt1 western blotting, run the same samples for GAPDH, and re-performed the quantitative analysis for Fig. 1A, 1G, 2C, and S7A. We did so for Fig. 2A, 5A, 5C, and S8A as well.
      21. Figure S15. Figure S15B and the left top panel in the source for Fig. S15 Authors’ statements Molecular weight markers were erroneously marked in all four panels in the original source data. Authors did not scrutinize paring pictures that showed positions of the markers. The markers were corrected in the revision. Conclusion Erroneous marking of molecular weights was appropriately corrected in the revision. Response 20. We re-performed anti-Hes5 western blotting, and confirmed the height of the bands of Hes5 and size markers (revised Fig. S15B and E).
      22. Figure S15. Figure S15BE and top two panels in the source for Fig. S15 (Hes5) Authors’ statements Bands for Hes5 (S15BE) and Gapdh (S15B) were erroneously marked in the original source data, and were corrected in the revision. Concern The concern is identical to concern #13. The indicated molecular weight of Hes5 (~35 kDa) is much different from it predicted molecular weight of 18 kDa. Conclusion Additional analyses are required. Erroneous marking of bands for Hes5 and Gapdh were corrected in the revision, but identity of Hes5 should be revealed. Response 21. As we mentioned in our response 20, we re-performed anti-Hes5 western blotting, and confirmed that the height of the bands of Hes5 is between 20 and 25 kDa (revised source file of Fig. S15B and E).
      23. Results for Figure 6FG (Hes5 expression after RG108 treatment) Concern In a section for “RG108 recovers Hes5 expression” in Results, authors state “it (Hes5) was diminished by RG108 treatment (Fig. 6F and G)”. This should be “enhanced” or “upregulated”. Conclusion The error should be corrected. Response 22. We corrected “diminished” to “up-regulated” in the text (Page 10, Line 13). 4th Editorial Decision 11 January 2019 Thank you for submitting your newly revised manuscript to EMBO Molecular Medicine. We have now carefully evaluated the figures along with the source data provided as well as the reply to the RIC letter that you kindly provided. While in principle, we are supportive of publication, still a few items remain to be corrected and some have to be added as follows: 1) Figures vs. source data: please fix!
      24. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 18 Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q
      25. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples I look forward to receiving a new revised version of your manuscript as soon as possible. 4th Revision - authors' response 1 March 2019 We are most grateful to you for your supportive comments on our manuscript. We revised Figures, Source data and manuscript according to the comment 1) 2) 5) and 6), and added created Author checklist, The Paper Explained, Synopsis, and Visual abstract in response to the comments 3) 4) 7) 8) and 9). Detailed responses are shown below. 1) Figures vs. source data: please fix!
      26. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q Response: In accordance to your instruction, we revised the Figures so that they match the source data, by removing any manipulation of angle and contrast of the images.
      27. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples Response: As you pointed out we had a mistake to label the data. We changed the label in the source of Appendix Figure S3B_GAPDH from “cerebellum” to “cortex”. USEFUL LINKS FOR COMPLETING THIS FORM http://www.antibodypedia.com http://1degreebio.org http://www.equator-network.org/reporting-guidelines/improving-bioscience-research-reporting-the-arrive-guidelines-for-reporting-http://grants.nih.gov/grants/olaw/olaw.htm http://www.mrc.ac.uk/Ourresearch/Ethicsresearchguidance/Useofanimals/index.htm http://ClinicalTrials.gov http://www.consort-statement.org http://www.consort-statement.org/checklists/view/32-consort/66-title ! http://www.equator-network.org/reporting-guidelines/reporting-recommendations-for-tumour-marker-prognostic-studies-! http://datadryad.org ! http://figshare.com ! http://www.ncbi.nlm.nih.gov/gap ! http://www.ebi.ac.uk/ega http://biomodels.net/ http://biomodels.net/miriam/ ! http://jjj.biochem.sun.ac.za ! http://oba.od.nih.gov/biosecurity/biosecurity_documents.html ! http://www.selectagents.gov/ ! ! ! ! ! " common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; " are tests one-sided or two-sided? " are there adjustments for multiple comparisons? " exact statistical test results, e.g., P values = x but not P values < x; " definition of ‘center values’ as median or average; " definition of error bars as s.d. or s.e.m. 1.a. How was the sample size chosen to ensure adequate power to detect a pre-specified effect size? 1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
      28. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria preestablished?
      29. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe. For animal studies, include a statement about randomization even if no randomization was used. 4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. 4.b. For animal studies, include a statement about blinding even if no blinding was done
      30. For every figure, are statistical tests justified as appropriate? Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Is there an estimate of variation within each group of data? Is the variance similar between the groups that are being statistically compared? Manuscript Number: EMM-2017-08547 EMBO PRESS A- Figures Reporting Checklist For Life Sciences Articles (Rev. June 2017) This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal’s authorship guidelines in preparing your manuscript. PLEASE NOTE THAT THIS CHECKLIST WILL BE PUBLISHED ALONGSIDE YOUR PAPER Journal Submitted to: EMBO molecular medicine Corresponding Author Name: Masahisa Katsuno a statement of how many times the experiment shown was independently replicated in the laboratory. Any descriptions too long for the figure legend should be included in the methods section and/or with the source data. In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects. definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.). Please fill out these boxes # (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name). C- Reagents B- Statistics and general methods the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
      31. Data the data were obtained and processed according to the field’s best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:
      32. Captions The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation. YOU MUST COMPLETE ALL CELLS WITH A PINK BACKGROUND # Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). NA We randomly allocated the mice to each treatment group (Page 15). We randomly allocated the mice to each treatment group (Page 15). The investigater blindly performed the behavior tests (Page 15). The investigater blindly performed the behavior tests (Page 15). Yes. We described the statistical methods in each legend. Yes. We confirmed the normality of data. Yes. We tested them using ANOVA. Yes. We confirmed the variance was similar between groups.
      33. To show that antibodies were profiled for use in the system under study (assay and species), provide a citation, catalog number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right).
      34. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination.
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      36. Report species, strain, gender, age of animals and genetic modification status where applicable. Please detail housing and husbandry conditions and the source of animals.
      37. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the committee(s) approving the experiments.
      38. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, 2010) to ensure that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.
      39. Identify the committee(s) approving the study protocol.
      40. Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
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      44. For phase II and III randomized controlled trials, please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission. See author guidelines, under ‘Reporting Guidelines’. Please confirm you have submitted this list.
      45. For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right). See author guidelines, under ‘Reporting Guidelines’. Please confirm you have followed these guidelines. 18: Provide a “Data Availability” section at the end of the Materials & Methods, listing the accession codes for data generated in this study and deposited in a public database (e.g. RNA-Seq data: Gene Expression Omnibus GSE39462, Proteomics data: PRIDE PXD000208 etc.) Please refer to our author guidelines for ‘Data Deposition’. Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Functional genomics data e. Proteomics and molecular interactions
      46. Deposition is strongly recommended for any datasets that are central and integral to the study; please consider the journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right).
      47. Access to human clinical and genomic datasets should be provided with as few restrictions as possible while respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public accesscontrolled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right).
      48. Computational models that are central and integral to a study should be shared without restrictions and provided in a machine-readable form. The relevant accession numbers or links should be provided. When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.
      49. Could your study fall under dual use research restrictions? Please check biosecurity documents (see link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could. F- Data Accessibility D- Animal Models E- Human Subjects We confirmed the guideline. G- Dual use research of concern We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page16-17 (section of Immunoblotting, and Histology and immunohistochemistry in the Material and Method). We described the details of cell lines on Page 6,8,9,and 22 (section of Result and Material and Method). We tested mycoplasma contamination. We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). NA NA NA NA NA NA NA NA
    3. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 1 DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5 Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, Masahisa Katsuno Review timeline: Submission date: 1 October 2017 Editorial Decision: 13 November 2017 Revision received: 27 February 2018 Editorial Decision: 6 April 2018 Revision received: 4 May 2018 Extra communication: 17 May 2018 Editorial Decision: 12 June 2018 Revision received: 7 January 2019 Editorial Decision: 11 Jannuary 2019 Revision received: 1 March 2019 Accepted: 1 March 2019 Editor: Céline Carret Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 13 November 2017 Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the two referees whom we asked to evaluate your manuscript. As you will see from the reports below, the referees find the data intriguing. Unfortunately, at this stage the work needs to be considerably strengthen to be more conclusive, and both referees provide good suggestions for that. Additional controls and experiments and adding mechanistic understanding seem to be required for the paper to be further evaluated in EMBO Molecular Medicine. Given that the referees find the message interesting, we would be willing to consider a revised manuscript with the understanding that the referee concerns must be fully addressed and that acceptance of the manuscript would entail a second round of review. I should remind you that EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. I realize that addressing the referees' comments in full would involve a lot of additional experimental work and resources and I am uncertain whether you will be able (or willing) to return a revised manuscript within the 3 months deadline. I would also understand your decision if you chose to rather seek rapid publication elsewhere at this stage. I look forward to seeing a revised form of your manuscript as soon as possible. Please read below for important editorial formatting and consult our author's guidelines for proper formatting of your revised article for EMBO Molecular Medicine. Should you find that the requested revisions are not feasible within the constraints outlined here and choose, therefore, to submit your paper elsewhere, we would welcome a message to this effect. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 2 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): Better control should be used as detailed to Authors Referee #1 (Remarks for Author): The study by Kondo al coll. Reports that the toxicity of the mutant AR responsible for SBMA could be ascribed to altered DNA methylation, and that this alteration could be reverted by the inhibition of this mechanisms using DNA methylation inhibitor such as RG108. In addition, the Authors have demonstrated that the hyper-methylation of the Hes5 gene correlates with the AR-97Q toxicity and that overexpression of Hes5 rescues from SBMA phenotype in cell models of the disease. While the study is potentially interesting, several points must be addressed in order to definitely prove that DNA methylation is relevant in SBMA. Major points:

      1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice.
      2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses.
      3. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motorneurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT.
      4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript
      5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells?
      6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108 EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 3
      7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers.
      8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice?
      9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR- 24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Minor Points
      10. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR.
      11. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Referee #2 (Comments on Novelty/Model System for Author): See comments to authors for 1 and 4. One issue is that validation in the NSC transformed cell line model is not sufficient. Referee #2 (Remarks for Author): In the Kondo et al. paper, a role for altered methylation in the polyglutamine disease SBMA is sought. The authors show Dnmt1 is upregulated within spinal motor neurons and suppression by siRNA or by a chemical inhibitor RG108 can suppress neuronal cell death. The authors translated the inhibitor into a pre-clinical mouse model and found evidence for suppression of SBMA neurodegenerative phenotypes. Intriguingly, this occurred downstream of polyglutamine aggregation, as aggregation was not affected. The authors show that cell viability in a NSC cell model of SBMA enabled by RG108 treatment depends on Hes5 expression, and that re-expression of Hes5 is sufficient to rescue viability. While the topic of methylation in neurodegeneration has not been addressed much, making the work intriguing, there are a number of problems with the investigation that undermine one's confidence in the results. The authors' explanation for which genes are affected by altered methylation is very superficial, and what Hes5 is doing is entirely unclear. As the validation of Hes5 takes place in only the transformed cell line model of SBMA, it seems rather premature to implicate Hes5 without providing a more thorough understanding of what it is doing. More concrete data is needed to balance what appears at times as speculation. Specific concerns that should be addressed to strengthen the manuscript are as follows: 1) The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. 2) A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and reviewers) can determine if the conclusions are valid. The problematic Figures are as follows:
      12. Fig 1C
      13. Fig 1D
      14. Fig 1E, EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 4
      15. Fig 1F
      16. Fig 2D
      17. Fig 3H
      18. Fig 5H
      19. Fig 6F
      20. Fig S1
      21. Fig S2
      22. Fig S3
      23. Fig S4
      24. Fig S5 3) All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. 4) As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Minor points: -There remain quite a few grammatical errors and typos, the authors should review the manuscript for these -The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? -Can the authors kindly provide the rationale for choosing RG108? -Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. -Figures 3A-D are missing significance labels -Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. 1st Revision - authors' response 27 February 2018 Referee #1: Major points: Point 1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice. Response 1: We thank the Referee for this important suggestion. In response to the proposal, we performed RT-qPCR of Dnmt1, Dnmt3a, and Dnmt3b using spinal cord samples from each group of mice (Fig. 1C). This analysis revealed that Dnmt1 mRNA level was up-regulated in AR-97Q mice as well as protein level. Point 2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 5 Response 2: In response to the Referee’s comment, we added the data of AR-24Q mice in Fig. 1A, 1B, 1C, 1D, 1E, 5I, and 5J, so that we excluded the influence of normal AR to the protein level and mRNA level of Dnmt1, Dnmt3a, Dnmt3b, and Hes5. Point 3-1. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. Response 3-1: We changed the term “expression” to “protein level” or “level” for the part of manuscript describing western blotting. In order to evaluate the gene expression level of Dnmt1, we performed RT-qPCR of NSC24Q and NSC97Q cells, and confirmed that Dnmt1 mRNA is expressed at a higher level in NSC97Q compared with NSC24Q (Fig. 2B). Point 3-2. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motor neurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. Response 3-2: We changed the sentence according to Referee’s suggestion (Page 7, Line 1-2). Point 3-3. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT. Response 3-3: To address the comment, we added the data comparing protein levels of Dnmts and Dnmt1 mRNA level in NSC97Q with or without DHT treatment (Fig. 2C and D). Moreover, we analyzed the influence of Dnmts knockdown on the cell viability of DHT-untreatd NSC97Q cells (Fig. 2F). Furthermore we evaluate the effect of RG108 to NSC97Q cells without DHT (Fig. 2I). The results of these additional experiments showed that Dnmt1 is not up-regulated in DHTuntreated NSC97Q cells, and that neither siRNA-mediated knockdown of Dnmts nor RG108 treatment alters the cell viability of NSC97Q cells without DHT treatment. Point 4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript. Response 4: According the Referee’s suggestion, we added the data of WST-8 assay of DHTtreated NSC24Q cells with siRNA-mediated knockdown of Dnmts (Supplementary Fig. 9) and RG108 treatment to DHT-treated NSC24Q (Fig. 2H and J). Both knockdown of Dnmts and RG108 had no effect on the cell viability of NSC24Q with DHT treatment. Point 5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells? Response 5: The DNA methylation array analysis we utilized was developed for human materials, but could not be applied to mouse samples at the time we performed experiments. Therefore we analyzed the samples of SH-SY5Y, human neuronal cells. In order to confirm that the phenomena observed in human cells are reproduced in murine cells, we performed all the experiments using cell samples derived from SH-SY5Y (Fig. 5 A, 5B, 5C, 5D, 5F, 5G, and 5H; Supplementary Fig. 12; Fig. 6A and 6B) and those from NSC34 (Fig. 2A and 2C; Supplementary Fig.13 and Fig. 6 C and 6D) in parallel. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 6 Point 6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108. Response 6: Our preliminary experiments revealed that Dnmt1 protein level was already elevated in the spinal cord of 6-week-old AR-97Q mouse (Figure for Referee shown below). This is the reason why we decided to start the brain injection treatment with RG108 to 6-week-old age mice. Point 7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers. Response 7: We changed the sentence to “DNA methylation array analysis using these cells revealed that DNA methylation of CpG islands is intensified in several genes. However, total DNA methylation level was not altered between SH24Q and SH97Q (Supplementary Fig. 11).” (Page 9, Line 7-9) Point 8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice? Response 8: To address this issue, we performed western blot using nuclear and cytoplasmic fraction of spinal cord lysate obtained from wild-type and AR97Q mice. Both in wild-type and AR97Q mice, Hes5 showed cytoplasm-dominant localization (Figure for Referee shown below). Although we cannot exclude the possibility that nuclear localization of Hes5 is somehow impaired in AR97Q mice, the western blot indicates that Hes5 is down-regulated both in nucleus and cytoplasm in SBMA model mouse, presumably resulting in the faint nuclear staining in the immunohistochemistry of the AR97Q mice. Point 9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR-24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Response 9: According to the comment, we added the data comparing DHT(-) and DHT(+) as shown in the Table for Referee shown below. As AR aggregation is not detectable in NSC97Q cell without DHT treatment, we did not perform the counterpart experiment for Fig. 7G and H. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 7 The data Referee pointed The corresponding data in the revise version Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig6. B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Minor Points Point 1. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR. Response: We are thankful to the suggestion. We changed RT-PCR to RT-qPCR throughout the manuscript. Point 2. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Response: We changed the term from “Wild” to “Wild-type”. Referee #2 Point 1. The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. Response 1: We thank the Referee for pointing this important issue. We now confirmed that relative mRNA level of human AR was not altered by RG108 treatment (Fig. 4E). This data indicated that RG108 had does not suppress AR transgene expression. Point 2. A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and Referees) can determine if the conclusions are valid. The problematic Figures are as follows:
      25. Fig 1C
      26. Fig 1D
      27. Fig 1E,
      28. Fig 1F
      29. Fig 2D
      30. Fig 3H
      31. Fig 5H
      32. Fig 6F
      33. Fig S1
      34. Fig S2
      35. Fig S3
      36. Fig S4
      37. Fig S5 Response 2: In response to the comment, we quantified the data as described below. Furthermore, we described the number of samples analyzed for quantitation in the figure legends and the Material and methods section (Page 17, Line 24 to Page 18 Line 3). EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 8 The data Referee pointed Quantification of data in the revise version Fig. 1C-E (now Fig. 1D) Fig. 1E Fig. 1F Mentioned in the manuscript (Page 5, Line 23-24) Fig. 2D (now Supplementary Fig. 7) Supplementary Fig. 7 Fig. 3H Fig. 3I Fig. 5H (now Fig. 5I) Fig. 5I Fig. 6F Fig. 6G Supplementary Fig. 1 Supplementary Fig. 1 Supplementary Fig. 2 Supplementary Fig. 2 Supplementary Fig. 3 Supplementary Fig. 3 Supplementary Fig. 4 Supplementary Fig. 4 Supplementary Fig. 5 (now Supplementary Fig. 6) Supplementary Fig. 6 Point 3. All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. Response 3: We added the data of cell model experiments without DHT treatment as shown in Table for Referee shown below. The figure in the initial submission The data including DHT-untreated cells in the revised version Fig. 2A Fig. 2C Fig. 2C (now Fig. 2E) Fig. 2F Fig. 2E (now Fig. 2G) Fig. 2I Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig. 6B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Point 4. As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Response 4: In order to explore the role of Hes5 in SBMA, we compared the Hes5 protein and mRNA levels in primary cortical neurons expressing AR24Q and AR97Q using lentivirus infection. AR97Q with DHT reduced the levels of Hes5 both in western blot and RT-qPCR in the primary cortical neurons as shown in NSC97Q (Fig. 8F, G). Furthermore, we confirmed that Hes5 reduction was observed in DHT-treated primary motor neurons expressing AR97Q compared with those expressing AR24Q (Fig. 8H). To further clarify the molecular basis for the beneficial effect of Hes5 in SBMA, we investigated the protein levels of key molecules in SBMA pathogenesis, such as heat shock factor-1 (HSF1), phosphorylated IkBa and Smad2, in NSC34 cells in which Hes5 is depleted. We found that phosphorylation of Smad2 is substantially down-regulated by siRNA-mediated knockdown of Hes5 despite Smad2 protein levels are not altered (Fig. 8A-C). To strengthen the data, we performed additional experiments. As shown in Fig. 8D, pSmad2 was down-regulated in DHT-treated NSC97Q cells compared with DHT-untreated NSC97Q (Fig. 8D). Moreover Hes5 over-expression induced up-regulation of pSmad2 both in NSC97Q and primary cortical neurons (Fig. 8E, I), indicating that Hes5 protects neurons from the toxic insults of polyglutamine-expanded AR via activation of Smad pathway. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 9 Minor points: Point 1. There remain quite a few grammatical errors and typos, the authors should review the manuscript for these Response 1: In response to this comment, our manuscript was proofread by Native speakers of English in Springer Nature Author Services. Point 2. The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? Response 2: In response to the comments, we performed quantitative analysis of Dnmt1 and 5mC in muscle. As shown in Supplementary Fig. 1A, the subcellular localization of Dnmt1 is exclusively in the nucleus. Quantitative analysis confirmed that nuclear levels of Dnmt1 are not altered in AR-97Q mouse (Supplementary Fig. 1B). Furthermore, the level of 5mc was also unaffected in the skeletal muscle of AR-97Q mice (Supplementary Fig. 5A and B). Point 3. Can the authors kindly provide the rationale for choosing RG108? Response 3: A previous study indicated that RG108 was safe and effective in a mouse model of amyotrophic lateral sclerosis (Chestnut BA et al, J Neurosci 2011).This work was also helpful for us to determine the concentration of the agent. Furthermore our preliminary data demonstrated that RG108 improved the viability of SBMA model cells. These findings made us to choose RG108. Point 4. Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. Response 4: Unfortunately, we could not perform blood counts of mice. Therefore, we deleted our description claiming that DNMT inhibitor may be useful in humans (Page 13). Point 5. Figures 3A-D are missing significance labels Response 5: We added the significance labels in Fig. 3A-D. Point 6. Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. Response 6: According to the comment, we explained the backbone and promoter information of Hes5 expression vector and mock vector in the Methods (Page 19, Line 8-9). 2nd Editorial Decision 6 April 2018 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending minor editorial amendments. Please submit your revised manuscript within two weeks. I look forward to seeing a revised form of your manuscript as soon as possible. I look forward to reading a new revised version of your manuscript as soon as possible. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 10 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): The Authors have improved he models used Referee #1 (Remarks for Author): All points were correctly addressed by the Authors Referee #2 (Remarks for Author): The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. 2nd Revision - authors' response 4 May 2018 Referee #1: All points were correctly addressed by the Authors. Response: We are truly grateful to you for your review of our work. Referee #2 The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. Response: We are so grateful to you for the constructive evaluation on our revision. We changed the sentence of conclusions using “possibly” according to the comments (Page 2, Line14). Communication concerning data integrity 17 May 2018 Thank you for sending us the source data for your revised manuscript " DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" at EMBO Molecular Medicine, and for your patience while we evaluated these additional information. We have carefully checked your figures and source data against one another and have come across several inconsistencies that necessitate further investigation. As you may know, the journal classifies image aberrations into three levels (http://embomolmed.embopress.org/classifying-image-aberrations), and we have classified the issues found in your figures collectively as a serious level I/borderline level II. In line with journal policies, this requires that we involve your research institution to provide an opportunity for quality control and investigation at the institutional level. In our experience, an institutional investigation can help greatly in identifying the causes of the apparent aberrations, as institutions can directly review lab books and interview the authors. It is also in your best interest to clarify the issues in a transparent manner with your employer. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 11 We would kindly invite you to let us know who would be the appropriate colleague in charge of research integrity to be contacted at your research institution. I look forward to hearing back form you very soon.

      RESPONSE FROM INSTITUTION AFTER INSTITUTIONAL INVESTIGATION In response to your request, we went through the manuscript by Dr. Masahisa Katsuno and checked the figures at the Ad Hoc Committee of the Research Integrity Committee (RIC) of Nagoya University. As a consequence, we found various mistakes in the figures which should be corrected or reanalyzed. The RIC concluded that the current manuscript does not stand as it is, and recommends the EMBO Molecular Medicine to suspend its publication. 3rd Editorial Decision 12 June 2018 Your manuscript #EMM-2017-08547-V3, entitled "DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" by Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, and Masahisa Katsuno has been withdrawn following recommendations of the Research Integrity Committee of Nagoya University who conducted a formal investigation. Should you be able to fix all issues found by the committee, with formal committee agreement that all issues have been fixed, and as long as that the study remains timely and of interest at the time of submission, we would not be opposed to evaluate it once more for publication as if it was an initial submission. 3rd Revision - authors' response 7 January 2019 CONFIRMATION FROM THE INSTITUTE AFTER AUTHORS PERFORMED REVISION This letter is concerning the manuscript titled ‘DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5’ y Kondo et al (EMM-2017-08547 V4). The research Integrity Committee of Nagoya University confirmed that the authors made revisions in response to comments and concerns by RIC. We agree that the journal reevaluates the appropriateness of the revisions. AUTHOR’S RESPONSE Ad Hoc Committee of the Research Integrity Committee (RIC)

      1. Figure 1. Fig. 1A and the right bottom panel in the source (Dntm3b) Authors’ statements They marked lanes 10, 11, and 12 for Dnmt3b in the original and revised source data. They noticed that the correct lanes were 7, 8, and 9, but have not fixed an error. Conclusion Authors need to fix an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 12 Response 1. We re-performed western blotting for Dnmt3b and confirmed the lanes which we showed in the main figure correspond to the lanes we marked in the revised source file of Fig. 1A. We also revised the quantitative analysis (Fig. 1B) due to the redo of western blot.
      2. Figure 1. Fig. 1G and the right middle panel in the source (GAPDH) Authors’ statements The excised bands for GAPDH in the original figure were wrong. In the revision, they claimed that the dense upper bands were correct GAPDH, and they revised Figure 1G and a frame in the source data accordingly. Concern The predicted molecular weight of GAPDH = 36 kDa. The original bands look correct and the revision looks wrong. Conclusion The authors need to recognize which bands are indeed GAPDH. Response 2. As we mentioned in our response 3 and 4, we re-performed western blotting for Dnmt1 using our original mouse skeletal muscle samples and run the same samples on a separate gel for GAPDH. We confirmed that GAPDH appears just below the molecular weight marker of 37 kDa. We revised Fig. 1G, and marked the right bands in the revised source file of Fig. 1G.
      3. Figure 1. Top two panels in the source for Fig. 1 (Dnmt1) Concern Mobilities of Dnmt1 are different between these two panels. Conclusion Additional analysis is required. The authors need to recognize which bands are indeed Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. Alternatively, the authors may immunoprecipitate Dnmt1 with anti-Dnmt1 antibody #1, and immunoblot the precipitated sample with anti-Dnmt1 antibody #2. Response 3. We noticed that the different results stemmed from the fact that we used antibodies with different Lot numbers. To solve this problem, we tested 3 kinds of antibodies (ab188453, Abcam; ab13537, Abcam; and sc20701, Santa Cruz), and found that ab188453 (1:1000) has the best ability to detect the bands. Using this antibody, we confirmed that Dnmt1 appears between 150 kDa and 250 kDa markers in the western blotting with the sample of NSC97Q cells treated with anti-Dnmt1 siRNA as a negative control (please refer the revised source file for Fig. 1A_Dnmt1). Based on the results of the additional western blotting, we changed the bands of Dnmt1 in Fig. 1A and 1G, and reperformed the quantitative analysis for Fig. 1B and 1H.
      4. Figure 1. Right top and middle panels in the source for Fig. 1 (GAPDH) Concern They first performed Western blotting (WB) with anti-Dnmt1 antibody (right top panel). Without stripping off the anti-Dnmt1 antibody, they reprobed the blot with anti-GAPDH antibody (right middle panel). Dnmt1 WB had many nonspecific bands, and some bands overlapped with GAPDH bands. They cannot estimate the amount of GAPDH without stripping off anti-Dnmt1 antibody. Conclusion Reanalysis is required. Strip off anti-Dnmt1 antibody or run samples on a separate gel for quantification of GAPDH. Response 4. As we mentioned in our Response 2, we re-performed western blotting for Dnmt1 with our original skeletal muscle samples of mice, and run the same samples on another gel for GAPDH to avoid the effects of stripping on re-probing. Based on the results of the additional western blotting, we changed the bands of GAPDH in Fig. 1A and 1G.
      5. Figure 2. Fig. 2A and the left 2nd panel in the source for Fig. 2AC (Dnmt3a) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 13 The molecular weight of Dnmt3a is more than 100 kDa, but looks less than 100 kDa. The RIC assumes that there was an error in the size marker. Conclusion Confirm the size markers and revise the source panel. Response 5. We re-performed western blotting using our original samples and confirmed that the bands of Dnmt3a appear above the marker of 100 kDa. Based on the results of the additional western blotting, we changed the bands of Dnmt3a and GAPDH in Fig. 2A, and re-performed the quantitative analysis.
      6. Figure 2. Fig. 2C and the left 3rd panel in the source for Fig. 2AC (Dnmt1) Authors’ statements Lanes 2-5, 6-9, and 10-13 were from triplicated experiments, and each group of bands looked similar. Dnmt1 in Fig. 2C were excised from lanes 4 and 5, but we inadvertently marked lanes 8 and 9 in the original source data. Conclusion Authors made an appropriate correction in the revised source data. Response 6. To clarify the alteration of Dnmt1 protein levels we re-performed anti-Dnmt1 western blotting using our original samples, and changed the bands of Dnmt1 in Fig. 2C and quantitative data.
      7. Figure 4. Fig. 4C and the left top panel in the source for Fig. 4 (GAPDH) Authors’ statements They could not find the source data for GAPDH in the original Fig. 4C. They thus used GAPDH bands of the same blot with different exposure in the source data. They excised GAPDH bands from the source data, and revised Fig. 4C. The first author looked for the original source data from Toronto, but the limited access to the source data disabled identification of the source data. After he came back to his laboratory, he has found the source data. Conclusion Authors need to make sure that GADPH bands in Fig. 4C and in the revised source data indeed represent correct samples. Response 7. The original data was lost while the first author moved from our lab to Toronto. We thus reperformed anti-AR western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of AR and GAPDH in Fig. 4C.
      8. Figure 4. Fig. 4C and the left bottom panel in the source for Fig. 4 (GAPDH) Concern The mark for 37 kDa does not match the position of a ladder. Conclusion Authors need to confirm the position of size markers. Response 8. As shown in the revised source file of Fig. 4C, we confirmed that GAPDH appears just below the molecular weight maker of 37 kDa.
      9. Figure 4. Fig. 4C and the two left panels in the source for Fig. 4 (Dnmt1 and GAPDH) Authors’ statements Lane marks for Dntm1 and GAPDH were wrong in the original and revised source data. Triplicated experiments were run on lanes 3-4, 5-6, and 7-8. Lanes 3-4 should have been marked, but lanes 2-3 were erroneously marked. Conclusion Authors need to correct an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 14 Response 9. As we mentioned in our response 7,we re-performed anti-AR western blotting, run the same samples on a separate gel for GAPDH (revised Fig. 4C), and re-performed the quantitative analysis (revised Fig. 4D).
      10. Figure 4. Fig. 4C and the top two panels in the source for Fig. 4 (Dnmt1 and Chat) Concern For both panels, triplicated experiments were run on lanes 3-4, 5-6, and 7-8. RG108 appears to similarly increase AR aggregates and Chat in lanes 4 and 8. Similarly, RG108 appears to similarly decrease AR aggregates and Chat lane 6. However, authors conclude that RG108 had no effect on AR aggregation (Fig. 4CD), but significantly increased Chat expression (Fig 4HI). In addition, AR aggregates in Fig. 4C do not appear to identical to the source data in the left upper panel. Conclusion Scrutinized reanalysis is required. The source data do not appear to support the authors' conclusions. Response 10. As explained in our response 7, we re-performed anti-AR western blotting, and confirmed that RG108 did not reduce the high molecular weight complex of AR (revised Fig. 4C). We also reperformed anti-Chat western blotting, and run the same samples for GAPDH. Based on the results of these additional experiments, we changed the bands of Chat and GAPDH (revised Fig. 4H), and reperformed the quantitative analysis (revised Fig. 4I).
      11. Figure 5. Fig. 5A and the right bottom panel in the source for Fig. 5AB (GAPDH) Authors’ statements In the original Fig. 5A, GAPDH bands were erroneously excised from non-specific bands. Correct GAPDH bands were excised in the revision. Conclusion The error was appropriately corrected in the revision. Response 11. To confirm Dnmt1 protein levels using the Dnmt1 antibody (ab188453) we re-performed western blotting using our original samples, and run the same samples for GAPDH to prevent that unstripped bands affect the detection of GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH (revised Fig. 5A), and re-performed the quantitative analysis (revised Fig. 5B).
      12. Figure 7. Fig. 7G and the two right panels in the source for Fig. 7EG (AR monomer and GAPDH) Authors’ statements The source data for AR monomer and GAPDH used in the original Fig. 7G could not be identified in an archive of electronic media. Authors instead found an image with longer exposure time of the same blot. In the revised Fig. 7G, authors excised AR monomer bands and GAPDH bands from the source data with longer exposure time in the revision. Conclusion The author should have archived the source data that they used in their manuscript. However, authors made an acceptable revision. Response 12. None (revision had been made).
      13. Figure 7. Fig. 7EG and the bottom two panels in the source for Fig. 7EG (GAPDH) Concern The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 13. As we mentioned in our response 12, we re-performed anti-GAPDH western blotting, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 15
      14. Figure 7. Figure 7EG and the left top panel in the source for Fig. 7EG (Hes5) Authors’ statements Authors were not aware of the difference between the predicted molecular weight (18 kDa) and the molecular weight of the stained band for Hes5 (~35 kDa). Concern The molecular weight of Hes5 is 18 kDa. The Hes5 bands marked by authors in Figs. 7EG, 8I, and S15BE were ~35 kDa and were much higher than 18 kDa. Datasheets of anti-Hes5 antibody by Novusbio and SantaCruz (M-104) show that Hes5 is stained at ~35 kDa, but the validity of these antibodies need to be scrutinized (Authors used the SantaCruz antibody). In addition, the position of the 50-kDa marker looks incorrect in the source data for Fig. 8I. Conclusion Additional analyses are required. Authors need to recognize the identity of Hes5 by performing Western blotting of cells that are knocked down for Hes5 and/or overexpress Hes5. Knockdown of Hes5 was already shown in S15B, but intensities of multiple bands were reduced. Confirmation by overexpression experiments is likely to be required. Response 14. We clarified that the height of HA-tagged Hes5 is between 20 and 25 kDa in anti-HA western blotting (attached below). We also found that the anti-Hes5 antibody we used in our original experiments (sc13859, Santa Cruz) detects bands between 20 and 25 kDa. Therefore, we re-trimmed such bands in Fig. 8C, 8E, 8F, and 8I, and re-performed the quantitative analysis of them. However, as our original results did not clearly detect Hes5 in Fig7B, 7E, S15B, and S15E, we re-performed additional western blotting of the original samples by shortening the electrophoresis time. According to the Committee’s comments, we run the same samples for western blotting of GAPDH to avoid the effects of stripping. Based on the results of these additional experiments, we changed the bands of Hes5 and GAPDH in Fig. 7B, 7E, S15B, and S15E, and re-performed the quantitative analysis of them. We confirmed that over-expression and knock-down of Hes5 were successfully done in each experiment.
      15. Figure S7. Fig. S7B and the left bottom panel in the source for Fig. S7 (GAPDH) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 16 The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 15. We re-analyzed the original blot, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa (revised source file of Fig S7B).
      16. Figure S7. Fig. S7AC (GAPDH) Concern Source data for GAPDH of Fig. S7AC are not indicated. Conclusion Show the relevant source data. Response 16. We re-performed anti-Dnmt1 western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH in Fig. S7A, and re-performed the quantitative analysis. We also add the result of anti- GAPDH re-probing of the blot shown in Fig. S7C, which was not included in our previous source file.
      17. Figure S7. Fig. S7B, and the right upper and left bottom panels in the source for Fig. S7 (Dnmt3a and GAPDH) Authors’ statements They changed the lane marks on the revised source data, but they claim that the original lane marks were correct. Conclusion Correct the lane marks again. Response 17. We re-marked the right bands in the source file of Fig. S7B.
      18. Figure S7. Fig. S7AC, and the upper two and right bottom panels in the source for Fig. S7 (Dnmt1 and Dnmt3a and Dnmt3b) Authors’ statements The authors marked lanes 5 and 6 in the original source data, but they should have been lanes 3 and
      19. The revised source data were marked correctly. They noticed that the lane marks on the right bottom panel for Dnmt3b should be Fig. S7C, but were erroneously marked as Fig. S7B. Conclusion Correct lanes were marked in the revision. Correct the tag from Fig. S7B to Fig. S7C. Response 18. As we mentioned in our responses 16 and 17, we re-performed western blotting for Fig. S7A, and re-marked the right bands in the source file of Fig. S7B and C, so that the source file corresponds to the Figures. We also corrected the label of Fig. S7C, which had been erroneously shown as “Fig. S7B”.
      20. Figure S7. Fig. S7AB and the left top panel in the source for Fig. S7 (Dnmt1) Concern The authors marked the strongest bands as Dnmt1 in the original and revised source data. However, knockdown of Dnmt1 did not reduce the signal intensity, although the authors showed that the signal intensities were reduced to about a half of controls. Dmnt1 marked in the source data for Figs. 1A, 2A, and 5A was slightly above a 150-kDA marker, whereas Dnmt1 marked in the source data for Fig. 1G was ~250-kDa. In Fig. S7A, the marked Dnmt1 is far above a 150-kDa marker. The RIC can recognize faint bands above the 150-kDa marker and below the strongest bands. Signal intensities of these faint bands were reduced by knockdown of Dnmt1. Conclusion Scrutinized reanalysis is required for Figs. 1A, 1G, 2A, 5A, and S7AB for identity of Dnmt1. This is repetition of concern #3. The authors need to identify Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 17 Response 19. As we mentioned in our responses 3, 6, and 7, we re-performed anti-Dnmt1 western blotting, run the same samples for GAPDH, and re-performed the quantitative analysis for Fig. 1A, 1G, 2C, and S7A. We did so for Fig. 2A, 5A, 5C, and S8A as well.
      21. Figure S15. Figure S15B and the left top panel in the source for Fig. S15 Authors’ statements Molecular weight markers were erroneously marked in all four panels in the original source data. Authors did not scrutinize paring pictures that showed positions of the markers. The markers were corrected in the revision. Conclusion Erroneous marking of molecular weights was appropriately corrected in the revision. Response 20. We re-performed anti-Hes5 western blotting, and confirmed the height of the bands of Hes5 and size markers (revised Fig. S15B and E).
      22. Figure S15. Figure S15BE and top two panels in the source for Fig. S15 (Hes5) Authors’ statements Bands for Hes5 (S15BE) and Gapdh (S15B) were erroneously marked in the original source data, and were corrected in the revision. Concern The concern is identical to concern #13. The indicated molecular weight of Hes5 (~35 kDa) is much different from it predicted molecular weight of 18 kDa. Conclusion Additional analyses are required. Erroneous marking of bands for Hes5 and Gapdh were corrected in the revision, but identity of Hes5 should be revealed. Response 21. As we mentioned in our response 20, we re-performed anti-Hes5 western blotting, and confirmed that the height of the bands of Hes5 is between 20 and 25 kDa (revised source file of Fig. S15B and E).
      23. Results for Figure 6FG (Hes5 expression after RG108 treatment) Concern In a section for “RG108 recovers Hes5 expression” in Results, authors state “it (Hes5) was diminished by RG108 treatment (Fig. 6F and G)”. This should be “enhanced” or “upregulated”. Conclusion The error should be corrected. Response 22. We corrected “diminished” to “up-regulated” in the text (Page 10, Line 13). 4th Editorial Decision 11 January 2019 Thank you for submitting your newly revised manuscript to EMBO Molecular Medicine. We have now carefully evaluated the figures along with the source data provided as well as the reply to the RIC letter that you kindly provided. While in principle, we are supportive of publication, still a few items remain to be corrected and some have to be added as follows: 1) Figures vs. source data: please fix!
      24. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 18 Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q
      25. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples I look forward to receiving a new revised version of your manuscript as soon as possible. 4th Revision - authors' response 1 March 2019 We are most grateful to you for your supportive comments on our manuscript. We revised Figures, Source data and manuscript according to the comment 1) 2) 5) and 6), and added created Author checklist, The Paper Explained, Synopsis, and Visual abstract in response to the comments 3) 4) 7) 8) and 9). Detailed responses are shown below. 1) Figures vs. source data: please fix!
      26. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q Response: In accordance to your instruction, we revised the Figures so that they match the source data, by removing any manipulation of angle and contrast of the images.
      27. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples Response: As you pointed out we had a mistake to label the data. We changed the label in the source of Appendix Figure S3B_GAPDH from “cerebellum” to “cortex”. USEFUL LINKS FOR COMPLETING THIS FORM http://www.antibodypedia.com http://1degreebio.org http://www.equator-network.org/reporting-guidelines/improving-bioscience-research-reporting-the-arrive-guidelines-for-reporting-http://grants.nih.gov/grants/olaw/olaw.htm http://www.mrc.ac.uk/Ourresearch/Ethicsresearchguidance/Useofanimals/index.htm http://ClinicalTrials.gov http://www.consort-statement.org http://www.consort-statement.org/checklists/view/32-consort/66-title ! http://www.equator-network.org/reporting-guidelines/reporting-recommendations-for-tumour-marker-prognostic-studies-! http://datadryad.org ! http://figshare.com ! http://www.ncbi.nlm.nih.gov/gap ! http://www.ebi.ac.uk/ega http://biomodels.net/ http://biomodels.net/miriam/ ! http://jjj.biochem.sun.ac.za ! http://oba.od.nih.gov/biosecurity/biosecurity_documents.html ! http://www.selectagents.gov/ ! ! ! ! ! " common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; " are tests one-sided or two-sided? " are there adjustments for multiple comparisons? " exact statistical test results, e.g., P values = x but not P values < x; " definition of ‘center values’ as median or average; " definition of error bars as s.d. or s.e.m. 1.a. How was the sample size chosen to ensure adequate power to detect a pre-specified effect size? 1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
      28. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria preestablished?
      29. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe. For animal studies, include a statement about randomization even if no randomization was used. 4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. 4.b. For animal studies, include a statement about blinding even if no blinding was done
      30. For every figure, are statistical tests justified as appropriate? Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Is there an estimate of variation within each group of data? Is the variance similar between the groups that are being statistically compared? Manuscript Number: EMM-2017-08547 EMBO PRESS A- Figures Reporting Checklist For Life Sciences Articles (Rev. June 2017) This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal’s authorship guidelines in preparing your manuscript. PLEASE NOTE THAT THIS CHECKLIST WILL BE PUBLISHED ALONGSIDE YOUR PAPER Journal Submitted to: EMBO molecular medicine Corresponding Author Name: Masahisa Katsuno a statement of how many times the experiment shown was independently replicated in the laboratory. Any descriptions too long for the figure legend should be included in the methods section and/or with the source data. In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects. definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.). Please fill out these boxes # (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name). C- Reagents B- Statistics and general methods the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
      31. Data the data were obtained and processed according to the field’s best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:
      32. Captions The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation. YOU MUST COMPLETE ALL CELLS WITH A PINK BACKGROUND # Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). NA We randomly allocated the mice to each treatment group (Page 15). We randomly allocated the mice to each treatment group (Page 15). The investigater blindly performed the behavior tests (Page 15). The investigater blindly performed the behavior tests (Page 15). Yes. We described the statistical methods in each legend. Yes. We confirmed the normality of data. Yes. We tested them using ANOVA. Yes. We confirmed the variance was similar between groups.
      33. To show that antibodies were profiled for use in the system under study (assay and species), provide a citation, catalog number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right).
      34. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination.
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      36. Report species, strain, gender, age of animals and genetic modification status where applicable. Please detail housing and husbandry conditions and the source of animals.
      37. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the committee(s) approving the experiments.
      38. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, 2010) to ensure that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.
      39. Identify the committee(s) approving the study protocol.
      40. Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
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      44. For phase II and III randomized controlled trials, please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission. See author guidelines, under ‘Reporting Guidelines’. Please confirm you have submitted this list.
      45. For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right). See author guidelines, under ‘Reporting Guidelines’. Please confirm you have followed these guidelines. 18: Provide a “Data Availability” section at the end of the Materials & Methods, listing the accession codes for data generated in this study and deposited in a public database (e.g. RNA-Seq data: Gene Expression Omnibus GSE39462, Proteomics data: PRIDE PXD000208 etc.) Please refer to our author guidelines for ‘Data Deposition’. Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Functional genomics data e. Proteomics and molecular interactions
      46. Deposition is strongly recommended for any datasets that are central and integral to the study; please consider the journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right).
      47. Access to human clinical and genomic datasets should be provided with as few restrictions as possible while respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public accesscontrolled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right).
      48. Computational models that are central and integral to a study should be shared without restrictions and provided in a machine-readable form. The relevant accession numbers or links should be provided. When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.
      49. Could your study fall under dual use research restrictions? Please check biosecurity documents (see link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could. F- Data Accessibility D- Animal Models E- Human Subjects We confirmed the guideline. G- Dual use research of concern We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page16-17 (section of Immunoblotting, and Histology and immunohistochemistry in the Material and Method). We described the details of cell lines on Page 6,8,9,and 22 (section of Result and Material and Method). We tested mycoplasma contamination. We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). NA NA NA NA NA NA NA NA
    4. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 1 DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5 Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, Masahisa Katsuno Review timeline: Submission date: 1 October 2017 Editorial Decision: 13 November 2017 Revision received: 27 February 2018 Editorial Decision: 6 April 2018 Revision received: 4 May 2018 Extra communication: 17 May 2018 Editorial Decision: 12 June 2018 Revision received: 7 January 2019 Editorial Decision: 11 Jannuary 2019 Revision received: 1 March 2019 Accepted: 1 March 2019 Editor: Céline Carret Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 13 November 2017 Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the two referees whom we asked to evaluate your manuscript. As you will see from the reports below, the referees find the data intriguing. Unfortunately, at this stage the work needs to be considerably strengthen to be more conclusive, and both referees provide good suggestions for that. Additional controls and experiments and adding mechanistic understanding seem to be required for the paper to be further evaluated in EMBO Molecular Medicine. Given that the referees find the message interesting, we would be willing to consider a revised manuscript with the understanding that the referee concerns must be fully addressed and that acceptance of the manuscript would entail a second round of review. I should remind you that EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. I realize that addressing the referees' comments in full would involve a lot of additional experimental work and resources and I am uncertain whether you will be able (or willing) to return a revised manuscript within the 3 months deadline. I would also understand your decision if you chose to rather seek rapid publication elsewhere at this stage. I look forward to seeing a revised form of your manuscript as soon as possible. Please read below for important editorial formatting and consult our author's guidelines for proper formatting of your revised article for EMBO Molecular Medicine. Should you find that the requested revisions are not feasible within the constraints outlined here and choose, therefore, to submit your paper elsewhere, we would welcome a message to this effect. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 2 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): Better control should be used as detailed to Authors Referee #1 (Remarks for Author): The study by Kondo al coll. Reports that the toxicity of the mutant AR responsible for SBMA could be ascribed to altered DNA methylation, and that this alteration could be reverted by the inhibition of this mechanisms using DNA methylation inhibitor such as RG108. In addition, the Authors have demonstrated that the hyper-methylation of the Hes5 gene correlates with the AR-97Q toxicity and that overexpression of Hes5 rescues from SBMA phenotype in cell models of the disease. While the study is potentially interesting, several points must be addressed in order to definitely prove that DNA methylation is relevant in SBMA. Major points:

      1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice.
      2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses.
      3. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motorneurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT.
      4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript
      5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells?
      6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108 EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 3
      7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers.
      8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice?
      9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR- 24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Minor Points
      10. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR.
      11. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Referee #2 (Comments on Novelty/Model System for Author): See comments to authors for 1 and 4. One issue is that validation in the NSC transformed cell line model is not sufficient. Referee #2 (Remarks for Author): In the Kondo et al. paper, a role for altered methylation in the polyglutamine disease SBMA is sought. The authors show Dnmt1 is upregulated within spinal motor neurons and suppression by siRNA or by a chemical inhibitor RG108 can suppress neuronal cell death. The authors translated the inhibitor into a pre-clinical mouse model and found evidence for suppression of SBMA neurodegenerative phenotypes. Intriguingly, this occurred downstream of polyglutamine aggregation, as aggregation was not affected. The authors show that cell viability in a NSC cell model of SBMA enabled by RG108 treatment depends on Hes5 expression, and that re-expression of Hes5 is sufficient to rescue viability. While the topic of methylation in neurodegeneration has not been addressed much, making the work intriguing, there are a number of problems with the investigation that undermine one's confidence in the results. The authors' explanation for which genes are affected by altered methylation is very superficial, and what Hes5 is doing is entirely unclear. As the validation of Hes5 takes place in only the transformed cell line model of SBMA, it seems rather premature to implicate Hes5 without providing a more thorough understanding of what it is doing. More concrete data is needed to balance what appears at times as speculation. Specific concerns that should be addressed to strengthen the manuscript are as follows: 1) The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. 2) A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and reviewers) can determine if the conclusions are valid. The problematic Figures are as follows:
      12. Fig 1C
      13. Fig 1D
      14. Fig 1E, EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 4
      15. Fig 1F
      16. Fig 2D
      17. Fig 3H
      18. Fig 5H
      19. Fig 6F
      20. Fig S1
      21. Fig S2
      22. Fig S3
      23. Fig S4
      24. Fig S5 3) All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. 4) As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Minor points: -There remain quite a few grammatical errors and typos, the authors should review the manuscript for these -The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? -Can the authors kindly provide the rationale for choosing RG108? -Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. -Figures 3A-D are missing significance labels -Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. 1st Revision - authors' response 27 February 2018 Referee #1: Major points: Point 1. Results, page 5, lines 13-14 and Figure 1. It is clear that the levels of the Dnmt1 protein, measured in WB or determined qualitatively in IF are increased. It will be of interest to determine whether this increase is due to an enhanced transcription in spinal cord or rather to a decreased turn over, which could be associated to the impact or the AR-97Q on the protein quality control system. In fact, AR-97Q may alter proteasome functions and this could reduce the overall clearance of a series of proteins processed via the proteasome. A RT-qPCR could be performed on RNA samples derived from spinal cord of wt and AR-97Q mice. Response 1: We thank the Referee for this important suggestion. In response to the proposal, we performed RT-qPCR of Dnmt1, Dnmt3a, and Dnmt3b using spinal cord samples from each group of mice (Fig. 1C). This analysis revealed that Dnmt1 mRNA level was up-regulated in AR-97Q mice as well as protein level. Point 2. Why the Authors decided to use the littermate non-transgenic mice, instead of the tg SBMA mice expressing the wt AR which have been developed by the same group in 2002? To exclude that the alterations observed (at least in basal conditions for the expression of Dnmts in Fig 1 and Hes5 in Fig 5H,I) are due to the presence of the overexpressed wtAR in motoneurons instead of the mutant AR-97Q, samples derived from the tg AR overexpressing mice must be included in these analyses. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 5 Response 2: In response to the Referee’s comment, we added the data of AR-24Q mice in Fig. 1A, 1B, 1C, 1D, 1E, 5I, and 5J, so that we excluded the influence of normal AR to the protein level and mRNA level of Dnmt1, Dnmt3a, Dnmt3b, and Hes5. Point 3-1. Page 6, line 20: The term "expression" is not correct. In WB (Figure 2 panels A and B), protein "levels" are detected. Changes in these levels may be associated to enhanced gene expression (measured determining RNA levels) and/or reduced degradation. As in point 1, a quantitative real time-PCR analysis is required to prove that enhanced Dnmt1 protein levels are linked to the transcriptional activation of the DNMT1 gene in response to the presence of testosterone activated AR-97Q in NSC34 cells. Response 3-1: We changed the term “expression” to “protein level” or “level” for the part of manuscript describing western blotting. In order to evaluate the gene expression level of Dnmt1, we performed RT-qPCR of NSC24Q and NSC97Q cells, and confirmed that Dnmt1 mRNA is expressed at a higher level in NSC97Q compared with NSC24Q (Fig. 2B). Point 3-2. In addition, please modify as follow: "....and Dnmt3b was not changed (Fig. 2A and B); the same phenomena were observed in spinal cord lysates of SBMA mice, as determined by western blot (see Fig. 1A and B)...." In fact, in page 6, line 23 the WB mentioned refers to spinal cord (which includes motor neurons along with many other cell types, e.g. astrocytes other neurons, etc.) and this analysis is reported in Fig. 1A and B. Fig. 2A and B mentioned here is related to the WB in NSC34 cells. Response 3-2: We changed the sentence according to Referee’s suggestion (Page 7, Line 1-2). Point 3-3. In all the experiments reported in Figure 2, it is required to use the control cells using NSC34 cells not treated with androgens (unactivated AR); in fact, these are stable transfected cells and the differences between NSC24Q and NCS97Q could be due to their clonal selection. The direct comparison between untreated and DHT treated cells will prove whether Dnmts levels are dependent on the AR-97Q toxicity triggered by androgens. No changes in Dnmt1 should be present in NSC97Q cells not exposed to DHT. Response 3-3: To address the comment, we added the data comparing protein levels of Dnmts and Dnmt1 mRNA level in NSC97Q with or without DHT treatment (Fig. 2C and D). Moreover, we analyzed the influence of Dnmts knockdown on the cell viability of DHT-untreatd NSC97Q cells (Fig. 2F). Furthermore we evaluate the effect of RG108 to NSC97Q cells without DHT (Fig. 2I). The results of these additional experiments showed that Dnmt1 is not up-regulated in DHTuntreated NSC97Q cells, and that neither siRNA-mediated knockdown of Dnmts nor RG108 treatment alters the cell viability of NSC97Q cells without DHT treatment. Point 4. Page 6, lines 24-24, Page 7, line 1 and Figure 2E: Is there an effect of Dnmt1 (and the other Dnmts) downregulation on the cell viability of testosterone treated NSC24Q? These data should be added into the supplementary material. The same is true for the experiments with RG108; the data obtained on wt cells (NSC24Q) should be included in the manuscript. Response 4: According the Referee’s suggestion, we added the data of WST-8 assay of DHTtreated NSC24Q cells with siRNA-mediated knockdown of Dnmts (Supplementary Fig. 9) and RG108 treatment to DHT-treated NSC24Q (Fig. 2H and J). Both knockdown of Dnmts and RG108 had no effect on the cell viability of NSC24Q with DHT treatment. Point 5. Page 8, Hes5 silencing in SH-SY5Y cells. Why all these experiments were conducted in neuroblastoma cells and the experiments reported in Fig 2 in NSC34 cells? Response 5: The DNA methylation array analysis we utilized was developed for human materials, but could not be applied to mouse samples at the time we performed experiments. Therefore we analyzed the samples of SH-SY5Y, human neuronal cells. In order to confirm that the phenomena observed in human cells are reproduced in murine cells, we performed all the experiments using cell samples derived from SH-SY5Y (Fig. 5 A, 5B, 5C, 5D, 5F, 5G, and 5H; Supplementary Fig. 12; Fig. 6A and 6B) and those from NSC34 (Fig. 2A and 2C; Supplementary Fig.13 and Fig. 6 C and 6D) in parallel. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 6 Point 6. Page 7, line 13: Why pre-symptomatic SBMA mice have been selected to start the treatment with RG108. Response 6: Our preliminary experiments revealed that Dnmt1 protein level was already elevated in the spinal cord of 6-week-old AR-97Q mouse (Figure for Referee shown below). This is the reason why we decided to start the brain injection treatment with RG108 to 6-week-old age mice. Point 7. Page 8, lines 20-23. Please re-phrase this paragraph, which is not clear to readers. Response 7: We changed the sentence to “DNA methylation array analysis using these cells revealed that DNA methylation of CpG islands is intensified in several genes. However, total DNA methylation level was not altered between SH24Q and SH97Q (Supplementary Fig. 11).” (Page 9, Line 7-9) Point 8. Page 9, line 16: Why Hes5 is diffused in the cytoplasm and nuclei of motorneurons of wt mice and it is only cytoplasmic in motorneurons of SBMA mice? Response 8: To address this issue, we performed western blot using nuclear and cytoplasmic fraction of spinal cord lysate obtained from wild-type and AR97Q mice. Both in wild-type and AR97Q mice, Hes5 showed cytoplasm-dominant localization (Figure for Referee shown below). Although we cannot exclude the possibility that nuclear localization of Hes5 is somehow impaired in AR97Q mice, the western blot indicates that Hes5 is down-regulated both in nucleus and cytoplasm in SBMA model mouse, presumably resulting in the faint nuclear staining in the immunohistochemistry of the AR97Q mice. Point 9. Figures 5A, 5D, 5G, 5E, 6B, 6D, 7A-H, as in the case of Fig 2 it is required to add as control the untreated NSC34 or SH-SY5Y stably expressing AR-97Q. Also in these cases the cells used are stably transfected cells and the differences observed may derive from clonal selection between AR-24Q and AR-97Q expressing cells. The analysis in cells not exposed to androgens in comparison the cells treated with DHT will allow to ascribe the variations of the levels of the proteins considered directly to the AR-97Q toxicity triggered by androgens. No changes in their levels should be noted in cells not exposed to DHT in all condition tested. Response 9: According to the comment, we added the data comparing DHT(-) and DHT(+) as shown in the Table for Referee shown below. As AR aggregation is not detectable in NSC97Q cell without DHT treatment, we did not perform the counterpart experiment for Fig. 7G and H. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 7 The data Referee pointed The corresponding data in the revise version Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig6. B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Minor Points Point 1. Page 9, line 3: the analysis performed is a quantitative analysis. RT-PCR usually refers to the qualitative PCR analysis performed on retro-transcribed RNA. It will be better to use RT-qPCR or Real Time-PCR. Response: We are thankful to the suggestion. We changed RT-PCR to RT-qPCR throughout the manuscript. Point 2. Please used the term "Wild type" instead of "Wild" in all figures in which these animals have been used. Response: We changed the term from “Wild” to “Wild-type”. Referee #2 Point 1. The AR97 mouse model shows over-expression of AR from an out-of-context promoter with little relationship to the normal regulation of AR. The authors need to exclude an effect upon AR transgene expression by RG108 as its mechanism of beneficial action. Response 1: We thank the Referee for pointing this important issue. We now confirmed that relative mRNA level of human AR was not altered by RG108 treatment (Fig. 4E). This data indicated that RG108 had does not suppress AR transgene expression. Point 2. A number of claims made by the authors are only supported by sample images without quantitation. All of these findings need to rigorously quantified and presented with statistical analysis. Also, the number of biological samples and number of frames used for image quantitation should be included in the Figure legends so that readers (and Referees) can determine if the conclusions are valid. The problematic Figures are as follows:
      25. Fig 1C
      26. Fig 1D
      27. Fig 1E,
      28. Fig 1F
      29. Fig 2D
      30. Fig 3H
      31. Fig 5H
      32. Fig 6F
      33. Fig S1
      34. Fig S2
      35. Fig S3
      36. Fig S4
      37. Fig S5 Response 2: In response to the comment, we quantified the data as described below. Furthermore, we described the number of samples analyzed for quantitation in the figure legends and the Material and methods section (Page 17, Line 24 to Page 18 Line 3). EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 8 The data Referee pointed Quantification of data in the revise version Fig. 1C-E (now Fig. 1D) Fig. 1E Fig. 1F Mentioned in the manuscript (Page 5, Line 23-24) Fig. 2D (now Supplementary Fig. 7) Supplementary Fig. 7 Fig. 3H Fig. 3I Fig. 5H (now Fig. 5I) Fig. 5I Fig. 6F Fig. 6G Supplementary Fig. 1 Supplementary Fig. 1 Supplementary Fig. 2 Supplementary Fig. 2 Supplementary Fig. 3 Supplementary Fig. 3 Supplementary Fig. 4 Supplementary Fig. 4 Supplementary Fig. 5 (now Supplementary Fig. 6) Supplementary Fig. 6 Point 3. All the cell model experiments use DHT+ conditions. Is the over-expression of Dnmt1 or suppression of Hes5 dependent on polyglutamine AR alone or must agonist treatment be included? These variations should be included as controls in such studies. Response 3: We added the data of cell model experiments without DHT treatment as shown in Table for Referee shown below. The figure in the initial submission The data including DHT-untreated cells in the revised version Fig. 2A Fig. 2C Fig. 2C (now Fig. 2E) Fig. 2F Fig. 2E (now Fig. 2G) Fig. 2I Fig. 5A Fig. 5C Fig. 5D (now Fig. 5F) Fig. 5G Fig. 5G (now Supplementary Fig. 13B) Supplementary Fig. 13D Fig. 5E (now Fig. 5H) Supplementary Fig. 12 Fig. 6B, D Supplementary Fig. 14A, B Fig. 7A-F Supplementary Fig. 15A-F Point 4. As noted above, the Hes5 connection to SBMA disease is tenuous at best, as the validation work is done in the NSC transformed cell line model, and the authors appear to have no idea whatsoever as the action of Hes5. Going further would make the work more convincing. Response 4: In order to explore the role of Hes5 in SBMA, we compared the Hes5 protein and mRNA levels in primary cortical neurons expressing AR24Q and AR97Q using lentivirus infection. AR97Q with DHT reduced the levels of Hes5 both in western blot and RT-qPCR in the primary cortical neurons as shown in NSC97Q (Fig. 8F, G). Furthermore, we confirmed that Hes5 reduction was observed in DHT-treated primary motor neurons expressing AR97Q compared with those expressing AR24Q (Fig. 8H). To further clarify the molecular basis for the beneficial effect of Hes5 in SBMA, we investigated the protein levels of key molecules in SBMA pathogenesis, such as heat shock factor-1 (HSF1), phosphorylated IkBa and Smad2, in NSC34 cells in which Hes5 is depleted. We found that phosphorylation of Smad2 is substantially down-regulated by siRNA-mediated knockdown of Hes5 despite Smad2 protein levels are not altered (Fig. 8A-C). To strengthen the data, we performed additional experiments. As shown in Fig. 8D, pSmad2 was down-regulated in DHT-treated NSC97Q cells compared with DHT-untreated NSC97Q (Fig. 8D). Moreover Hes5 over-expression induced up-regulation of pSmad2 both in NSC97Q and primary cortical neurons (Fig. 8E, I), indicating that Hes5 protects neurons from the toxic insults of polyglutamine-expanded AR via activation of Smad pathway. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 9 Minor points: Point 1. There remain quite a few grammatical errors and typos, the authors should review the manuscript for these Response 1: In response to this comment, our manuscript was proofread by Native speakers of English in Springer Nature Author Services. Point 2. The control for no over-expresssion of Dnmt1 in muscle tissue in Fig. 1G,H (and S1-3) is welcome, however the authors suggest localization of Dnmt1 may be most important. Can the authors provide any quantified evidence, such as subcellular localization or 5-methylcytosine levels, in muscle tissue? Response 2: In response to the comments, we performed quantitative analysis of Dnmt1 and 5mC in muscle. As shown in Supplementary Fig. 1A, the subcellular localization of Dnmt1 is exclusively in the nucleus. Quantitative analysis confirmed that nuclear levels of Dnmt1 are not altered in AR-97Q mouse (Supplementary Fig. 1B). Furthermore, the level of 5mc was also unaffected in the skeletal muscle of AR-97Q mice (Supplementary Fig. 5A and B). Point 3. Can the authors kindly provide the rationale for choosing RG108? Response 3: A previous study indicated that RG108 was safe and effective in a mouse model of amyotrophic lateral sclerosis (Chestnut BA et al, J Neurosci 2011).This work was also helpful for us to determine the concentration of the agent. Furthermore our preliminary data demonstrated that RG108 improved the viability of SBMA model cells. These findings made us to choose RG108. Point 4. Clinical inhibitors for DNMTs, including azacytidine and decitabine, have relatively high toxicity. Was the authors' hope with RG108 to more specifically target the specific DNMT affected? Can the authors provide any evidence of toxicity to blood counts in mice, as is found in patients? If these data are not available, the authors should qualify their statement suggesting DNMT inhibitors may be useful in humans; mice are in a sterile environment and DNMTs are typically administered for short periods of time. Response 4: Unfortunately, we could not perform blood counts of mice. Therefore, we deleted our description claiming that DNMT inhibitor may be useful in humans (Page 13). Point 5. Figures 3A-D are missing significance labels Response 5: We added the significance labels in Fig. 3A-D. Point 6. Please document the backbone and other characteristics (promoter?) of the Hes5 expression vector and mock vector. Response 6: According to the comment, we explained the backbone and promoter information of Hes5 expression vector and mock vector in the Methods (Page 19, Line 8-9). 2nd Editorial Decision 6 April 2018 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending minor editorial amendments. Please submit your revised manuscript within two weeks. I look forward to seeing a revised form of your manuscript as soon as possible. I look forward to reading a new revised version of your manuscript as soon as possible. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 10 * Reviewer's comments * Referee #1 (Comments on Novelty/Model System for Author): The Authors have improved he models used Referee #1 (Remarks for Author): All points were correctly addressed by the Authors Referee #2 (Remarks for Author): The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. 2nd Revision - authors' response 4 May 2018 Referee #1: All points were correctly addressed by the Authors. Response: We are truly grateful to you for your review of our work. Referee #2 The authors have done an excellent job of revising the manuscript and have addressed my main concerns. I am not entirely convinced however that their examination of the Smad2 pathway sufficiently distinguished between correlation and causation, and thus request that the authors temper their conclusions regarding Smad by rewriting sentences to include "may" or "possibly", instead of drawing definitive conclusions. Response: We are so grateful to you for the constructive evaluation on our revision. We changed the sentence of conclusions using “possibly” according to the comments (Page 2, Line14). Communication concerning data integrity 17 May 2018 Thank you for sending us the source data for your revised manuscript " DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" at EMBO Molecular Medicine, and for your patience while we evaluated these additional information. We have carefully checked your figures and source data against one another and have come across several inconsistencies that necessitate further investigation. As you may know, the journal classifies image aberrations into three levels (http://embomolmed.embopress.org/classifying-image-aberrations), and we have classified the issues found in your figures collectively as a serious level I/borderline level II. In line with journal policies, this requires that we involve your research institution to provide an opportunity for quality control and investigation at the institutional level. In our experience, an institutional investigation can help greatly in identifying the causes of the apparent aberrations, as institutions can directly review lab books and interview the authors. It is also in your best interest to clarify the issues in a transparent manner with your employer. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 11 We would kindly invite you to let us know who would be the appropriate colleague in charge of research integrity to be contacted at your research institution. I look forward to hearing back form you very soon.

      RESPONSE FROM INSTITUTION AFTER INSTITUTIONAL INVESTIGATION In response to your request, we went through the manuscript by Dr. Masahisa Katsuno and checked the figures at the Ad Hoc Committee of the Research Integrity Committee (RIC) of Nagoya University. As a consequence, we found various mistakes in the figures which should be corrected or reanalyzed. The RIC concluded that the current manuscript does not stand as it is, and recommends the EMBO Molecular Medicine to suspend its publication. 3rd Editorial Decision 12 June 2018 Your manuscript #EMM-2017-08547-V3, entitled "DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5" by Naohide Kondo, Genki Tohnai, Kentaro Sahashi, Madoka Iida, Mayumi Kataoka, Hideaki Nakatsuji, Yutaka Tsutsumi, Atsushi Hashizume, Hiroaki Adachi, Haruki Koike, Keiko Shinjo, Yutaka Kondo, Gen Sobue, and Masahisa Katsuno has been withdrawn following recommendations of the Research Integrity Committee of Nagoya University who conducted a formal investigation. Should you be able to fix all issues found by the committee, with formal committee agreement that all issues have been fixed, and as long as that the study remains timely and of interest at the time of submission, we would not be opposed to evaluate it once more for publication as if it was an initial submission. 3rd Revision - authors' response 7 January 2019 CONFIRMATION FROM THE INSTITUTE AFTER AUTHORS PERFORMED REVISION This letter is concerning the manuscript titled ‘DNA methylation inhibitor attenuates polyglutamineinduced neurodegeneration by regulating Hes5’ y Kondo et al (EMM-2017-08547 V4). The research Integrity Committee of Nagoya University confirmed that the authors made revisions in response to comments and concerns by RIC. We agree that the journal reevaluates the appropriateness of the revisions. AUTHOR’S RESPONSE Ad Hoc Committee of the Research Integrity Committee (RIC)

      1. Figure 1. Fig. 1A and the right bottom panel in the source (Dntm3b) Authors’ statements They marked lanes 10, 11, and 12 for Dnmt3b in the original and revised source data. They noticed that the correct lanes were 7, 8, and 9, but have not fixed an error. Conclusion Authors need to fix an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 12 Response 1. We re-performed western blotting for Dnmt3b and confirmed the lanes which we showed in the main figure correspond to the lanes we marked in the revised source file of Fig. 1A. We also revised the quantitative analysis (Fig. 1B) due to the redo of western blot.
      2. Figure 1. Fig. 1G and the right middle panel in the source (GAPDH) Authors’ statements The excised bands for GAPDH in the original figure were wrong. In the revision, they claimed that the dense upper bands were correct GAPDH, and they revised Figure 1G and a frame in the source data accordingly. Concern The predicted molecular weight of GAPDH = 36 kDa. The original bands look correct and the revision looks wrong. Conclusion The authors need to recognize which bands are indeed GAPDH. Response 2. As we mentioned in our response 3 and 4, we re-performed western blotting for Dnmt1 using our original mouse skeletal muscle samples and run the same samples on a separate gel for GAPDH. We confirmed that GAPDH appears just below the molecular weight marker of 37 kDa. We revised Fig. 1G, and marked the right bands in the revised source file of Fig. 1G.
      3. Figure 1. Top two panels in the source for Fig. 1 (Dnmt1) Concern Mobilities of Dnmt1 are different between these two panels. Conclusion Additional analysis is required. The authors need to recognize which bands are indeed Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. Alternatively, the authors may immunoprecipitate Dnmt1 with anti-Dnmt1 antibody #1, and immunoblot the precipitated sample with anti-Dnmt1 antibody #2. Response 3. We noticed that the different results stemmed from the fact that we used antibodies with different Lot numbers. To solve this problem, we tested 3 kinds of antibodies (ab188453, Abcam; ab13537, Abcam; and sc20701, Santa Cruz), and found that ab188453 (1:1000) has the best ability to detect the bands. Using this antibody, we confirmed that Dnmt1 appears between 150 kDa and 250 kDa markers in the western blotting with the sample of NSC97Q cells treated with anti-Dnmt1 siRNA as a negative control (please refer the revised source file for Fig. 1A_Dnmt1). Based on the results of the additional western blotting, we changed the bands of Dnmt1 in Fig. 1A and 1G, and reperformed the quantitative analysis for Fig. 1B and 1H.
      4. Figure 1. Right top and middle panels in the source for Fig. 1 (GAPDH) Concern They first performed Western blotting (WB) with anti-Dnmt1 antibody (right top panel). Without stripping off the anti-Dnmt1 antibody, they reprobed the blot with anti-GAPDH antibody (right middle panel). Dnmt1 WB had many nonspecific bands, and some bands overlapped with GAPDH bands. They cannot estimate the amount of GAPDH without stripping off anti-Dnmt1 antibody. Conclusion Reanalysis is required. Strip off anti-Dnmt1 antibody or run samples on a separate gel for quantification of GAPDH. Response 4. As we mentioned in our Response 2, we re-performed western blotting for Dnmt1 with our original skeletal muscle samples of mice, and run the same samples on another gel for GAPDH to avoid the effects of stripping on re-probing. Based on the results of the additional western blotting, we changed the bands of GAPDH in Fig. 1A and 1G.
      5. Figure 2. Fig. 2A and the left 2nd panel in the source for Fig. 2AC (Dnmt3a) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 13 The molecular weight of Dnmt3a is more than 100 kDa, but looks less than 100 kDa. The RIC assumes that there was an error in the size marker. Conclusion Confirm the size markers and revise the source panel. Response 5. We re-performed western blotting using our original samples and confirmed that the bands of Dnmt3a appear above the marker of 100 kDa. Based on the results of the additional western blotting, we changed the bands of Dnmt3a and GAPDH in Fig. 2A, and re-performed the quantitative analysis.
      6. Figure 2. Fig. 2C and the left 3rd panel in the source for Fig. 2AC (Dnmt1) Authors’ statements Lanes 2-5, 6-9, and 10-13 were from triplicated experiments, and each group of bands looked similar. Dnmt1 in Fig. 2C were excised from lanes 4 and 5, but we inadvertently marked lanes 8 and 9 in the original source data. Conclusion Authors made an appropriate correction in the revised source data. Response 6. To clarify the alteration of Dnmt1 protein levels we re-performed anti-Dnmt1 western blotting using our original samples, and changed the bands of Dnmt1 in Fig. 2C and quantitative data.
      7. Figure 4. Fig. 4C and the left top panel in the source for Fig. 4 (GAPDH) Authors’ statements They could not find the source data for GAPDH in the original Fig. 4C. They thus used GAPDH bands of the same blot with different exposure in the source data. They excised GAPDH bands from the source data, and revised Fig. 4C. The first author looked for the original source data from Toronto, but the limited access to the source data disabled identification of the source data. After he came back to his laboratory, he has found the source data. Conclusion Authors need to make sure that GADPH bands in Fig. 4C and in the revised source data indeed represent correct samples. Response 7. The original data was lost while the first author moved from our lab to Toronto. We thus reperformed anti-AR western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of AR and GAPDH in Fig. 4C.
      8. Figure 4. Fig. 4C and the left bottom panel in the source for Fig. 4 (GAPDH) Concern The mark for 37 kDa does not match the position of a ladder. Conclusion Authors need to confirm the position of size markers. Response 8. As shown in the revised source file of Fig. 4C, we confirmed that GAPDH appears just below the molecular weight maker of 37 kDa.
      9. Figure 4. Fig. 4C and the two left panels in the source for Fig. 4 (Dnmt1 and GAPDH) Authors’ statements Lane marks for Dntm1 and GAPDH were wrong in the original and revised source data. Triplicated experiments were run on lanes 3-4, 5-6, and 7-8. Lanes 3-4 should have been marked, but lanes 2-3 were erroneously marked. Conclusion Authors need to correct an error. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 14 Response 9. As we mentioned in our response 7,we re-performed anti-AR western blotting, run the same samples on a separate gel for GAPDH (revised Fig. 4C), and re-performed the quantitative analysis (revised Fig. 4D).
      10. Figure 4. Fig. 4C and the top two panels in the source for Fig. 4 (Dnmt1 and Chat) Concern For both panels, triplicated experiments were run on lanes 3-4, 5-6, and 7-8. RG108 appears to similarly increase AR aggregates and Chat in lanes 4 and 8. Similarly, RG108 appears to similarly decrease AR aggregates and Chat lane 6. However, authors conclude that RG108 had no effect on AR aggregation (Fig. 4CD), but significantly increased Chat expression (Fig 4HI). In addition, AR aggregates in Fig. 4C do not appear to identical to the source data in the left upper panel. Conclusion Scrutinized reanalysis is required. The source data do not appear to support the authors' conclusions. Response 10. As explained in our response 7, we re-performed anti-AR western blotting, and confirmed that RG108 did not reduce the high molecular weight complex of AR (revised Fig. 4C). We also reperformed anti-Chat western blotting, and run the same samples for GAPDH. Based on the results of these additional experiments, we changed the bands of Chat and GAPDH (revised Fig. 4H), and reperformed the quantitative analysis (revised Fig. 4I).
      11. Figure 5. Fig. 5A and the right bottom panel in the source for Fig. 5AB (GAPDH) Authors’ statements In the original Fig. 5A, GAPDH bands were erroneously excised from non-specific bands. Correct GAPDH bands were excised in the revision. Conclusion The error was appropriately corrected in the revision. Response 11. To confirm Dnmt1 protein levels using the Dnmt1 antibody (ab188453) we re-performed western blotting using our original samples, and run the same samples for GAPDH to prevent that unstripped bands affect the detection of GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH (revised Fig. 5A), and re-performed the quantitative analysis (revised Fig. 5B).
      12. Figure 7. Fig. 7G and the two right panels in the source for Fig. 7EG (AR monomer and GAPDH) Authors’ statements The source data for AR monomer and GAPDH used in the original Fig. 7G could not be identified in an archive of electronic media. Authors instead found an image with longer exposure time of the same blot. In the revised Fig. 7G, authors excised AR monomer bands and GAPDH bands from the source data with longer exposure time in the revision. Conclusion The author should have archived the source data that they used in their manuscript. However, authors made an acceptable revision. Response 12. None (revision had been made).
      13. Figure 7. Fig. 7EG and the bottom two panels in the source for Fig. 7EG (GAPDH) Concern The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 13. As we mentioned in our response 12, we re-performed anti-GAPDH western blotting, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 15
      14. Figure 7. Figure 7EG and the left top panel in the source for Fig. 7EG (Hes5) Authors’ statements Authors were not aware of the difference between the predicted molecular weight (18 kDa) and the molecular weight of the stained band for Hes5 (~35 kDa). Concern The molecular weight of Hes5 is 18 kDa. The Hes5 bands marked by authors in Figs. 7EG, 8I, and S15BE were ~35 kDa and were much higher than 18 kDa. Datasheets of anti-Hes5 antibody by Novusbio and SantaCruz (M-104) show that Hes5 is stained at ~35 kDa, but the validity of these antibodies need to be scrutinized (Authors used the SantaCruz antibody). In addition, the position of the 50-kDa marker looks incorrect in the source data for Fig. 8I. Conclusion Additional analyses are required. Authors need to recognize the identity of Hes5 by performing Western blotting of cells that are knocked down for Hes5 and/or overexpress Hes5. Knockdown of Hes5 was already shown in S15B, but intensities of multiple bands were reduced. Confirmation by overexpression experiments is likely to be required. Response 14. We clarified that the height of HA-tagged Hes5 is between 20 and 25 kDa in anti-HA western blotting (attached below). We also found that the anti-Hes5 antibody we used in our original experiments (sc13859, Santa Cruz) detects bands between 20 and 25 kDa. Therefore, we re-trimmed such bands in Fig. 8C, 8E, 8F, and 8I, and re-performed the quantitative analysis of them. However, as our original results did not clearly detect Hes5 in Fig7B, 7E, S15B, and S15E, we re-performed additional western blotting of the original samples by shortening the electrophoresis time. According to the Committee’s comments, we run the same samples for western blotting of GAPDH to avoid the effects of stripping. Based on the results of these additional experiments, we changed the bands of Hes5 and GAPDH in Fig. 7B, 7E, S15B, and S15E, and re-performed the quantitative analysis of them. We confirmed that over-expression and knock-down of Hes5 were successfully done in each experiment.
      15. Figure S7. Fig. S7B and the left bottom panel in the source for Fig. S7 (GAPDH) Concern EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 16 The molecular weight of GAPDH is 36 kDa. A marker for 37 kDa appears to be wrongly marked. Conclusion Authors need to scrutinize size markers throughout their source data. Response 15. We re-analyzed the original blot, and confirmed that GAPDH appears just below the molecular weight maker of 37 kDa (revised source file of Fig S7B).
      16. Figure S7. Fig. S7AC (GAPDH) Concern Source data for GAPDH of Fig. S7AC are not indicated. Conclusion Show the relevant source data. Response 16. We re-performed anti-Dnmt1 western blotting using our original samples, and run the same samples for GAPDH. Based on the results of the additional western blotting, we changed the bands of Dnmt1 and GAPDH in Fig. S7A, and re-performed the quantitative analysis. We also add the result of anti- GAPDH re-probing of the blot shown in Fig. S7C, which was not included in our previous source file.
      17. Figure S7. Fig. S7B, and the right upper and left bottom panels in the source for Fig. S7 (Dnmt3a and GAPDH) Authors’ statements They changed the lane marks on the revised source data, but they claim that the original lane marks were correct. Conclusion Correct the lane marks again. Response 17. We re-marked the right bands in the source file of Fig. S7B.
      18. Figure S7. Fig. S7AC, and the upper two and right bottom panels in the source for Fig. S7 (Dnmt1 and Dnmt3a and Dnmt3b) Authors’ statements The authors marked lanes 5 and 6 in the original source data, but they should have been lanes 3 and
      19. The revised source data were marked correctly. They noticed that the lane marks on the right bottom panel for Dnmt3b should be Fig. S7C, but were erroneously marked as Fig. S7B. Conclusion Correct lanes were marked in the revision. Correct the tag from Fig. S7B to Fig. S7C. Response 18. As we mentioned in our responses 16 and 17, we re-performed western blotting for Fig. S7A, and re-marked the right bands in the source file of Fig. S7B and C, so that the source file corresponds to the Figures. We also corrected the label of Fig. S7C, which had been erroneously shown as “Fig. S7B”.
      20. Figure S7. Fig. S7AB and the left top panel in the source for Fig. S7 (Dnmt1) Concern The authors marked the strongest bands as Dnmt1 in the original and revised source data. However, knockdown of Dnmt1 did not reduce the signal intensity, although the authors showed that the signal intensities were reduced to about a half of controls. Dmnt1 marked in the source data for Figs. 1A, 2A, and 5A was slightly above a 150-kDA marker, whereas Dnmt1 marked in the source data for Fig. 1G was ~250-kDa. In Fig. S7A, the marked Dnmt1 is far above a 150-kDa marker. The RIC can recognize faint bands above the 150-kDa marker and below the strongest bands. Signal intensities of these faint bands were reduced by knockdown of Dnmt1. Conclusion Scrutinized reanalysis is required for Figs. 1A, 1G, 2A, 5A, and S7AB for identity of Dnmt1. This is repetition of concern #3. The authors need to identify Dnmt1 by performing Western blotting of cells that are knocked down for Dnmt1 and/or overexpress Dnmt1. EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 17 Response 19. As we mentioned in our responses 3, 6, and 7, we re-performed anti-Dnmt1 western blotting, run the same samples for GAPDH, and re-performed the quantitative analysis for Fig. 1A, 1G, 2C, and S7A. We did so for Fig. 2A, 5A, 5C, and S8A as well.
      21. Figure S15. Figure S15B and the left top panel in the source for Fig. S15 Authors’ statements Molecular weight markers were erroneously marked in all four panels in the original source data. Authors did not scrutinize paring pictures that showed positions of the markers. The markers were corrected in the revision. Conclusion Erroneous marking of molecular weights was appropriately corrected in the revision. Response 20. We re-performed anti-Hes5 western blotting, and confirmed the height of the bands of Hes5 and size markers (revised Fig. S15B and E).
      22. Figure S15. Figure S15BE and top two panels in the source for Fig. S15 (Hes5) Authors’ statements Bands for Hes5 (S15BE) and Gapdh (S15B) were erroneously marked in the original source data, and were corrected in the revision. Concern The concern is identical to concern #13. The indicated molecular weight of Hes5 (~35 kDa) is much different from it predicted molecular weight of 18 kDa. Conclusion Additional analyses are required. Erroneous marking of bands for Hes5 and Gapdh were corrected in the revision, but identity of Hes5 should be revealed. Response 21. As we mentioned in our response 20, we re-performed anti-Hes5 western blotting, and confirmed that the height of the bands of Hes5 is between 20 and 25 kDa (revised source file of Fig. S15B and E).
      23. Results for Figure 6FG (Hes5 expression after RG108 treatment) Concern In a section for “RG108 recovers Hes5 expression” in Results, authors state “it (Hes5) was diminished by RG108 treatment (Fig. 6F and G)”. This should be “enhanced” or “upregulated”. Conclusion The error should be corrected. Response 22. We corrected “diminished” to “up-regulated” in the text (Page 10, Line 13). 4th Editorial Decision 11 January 2019 Thank you for submitting your newly revised manuscript to EMBO Molecular Medicine. We have now carefully evaluated the figures along with the source data provided as well as the reply to the RIC letter that you kindly provided. While in principle, we are supportive of publication, still a few items remain to be corrected and some have to be added as follows: 1) Figures vs. source data: please fix!
      24. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH EMBO Molecular Medicine - Peer Review Process File © European Molecular Biology Organization 18 Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q
      25. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples I look forward to receiving a new revised version of your manuscript as soon as possible. 4th Revision - authors' response 1 March 2019 We are most grateful to you for your supportive comments on our manuscript. We revised Figures, Source data and manuscript according to the comment 1) 2) 5) and 6), and added created Author checklist, The Paper Explained, Synopsis, and Visual abstract in response to the comments 3) 4) 7) 8) and 9). Detailed responses are shown below. 1) Figures vs. source data: please fix!
      26. the bands in the figure do not seem to be the same as in the source data in Figure 5C_Dnmt3b Figure 8A_Smad2 Figure 8D_Smad2 Figure 8F_Smad2 Figure 8I_GAPDH Appendix Figure S12_SH97Q (different exposures in figure that contradicts the appendix figure) both M and U Appendix FigureS13C_NSC97Q Response: In accordance to your instruction, we revised the Figures so that they match the source data, by removing any manipulation of angle and contrast of the images.
      27. Labelling mistake we believe in Appendix Figure S3B_GAPDH (labelled twice as such, for cerebellum), we believe that one of them matches the cortex samples Response: As you pointed out we had a mistake to label the data. We changed the label in the source of Appendix Figure S3B_GAPDH from “cerebellum” to “cortex”. USEFUL LINKS FOR COMPLETING THIS FORM http://www.antibodypedia.com http://1degreebio.org http://www.equator-network.org/reporting-guidelines/improving-bioscience-research-reporting-the-arrive-guidelines-for-reporting-http://grants.nih.gov/grants/olaw/olaw.htm http://www.mrc.ac.uk/Ourresearch/Ethicsresearchguidance/Useofanimals/index.htm http://ClinicalTrials.gov http://www.consort-statement.org http://www.consort-statement.org/checklists/view/32-consort/66-title ! http://www.equator-network.org/reporting-guidelines/reporting-recommendations-for-tumour-marker-prognostic-studies-! http://datadryad.org ! http://figshare.com ! http://www.ncbi.nlm.nih.gov/gap ! http://www.ebi.ac.uk/ega http://biomodels.net/ http://biomodels.net/miriam/ ! http://jjj.biochem.sun.ac.za ! http://oba.od.nih.gov/biosecurity/biosecurity_documents.html ! http://www.selectagents.gov/ ! ! ! ! ! " common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; " are tests one-sided or two-sided? " are there adjustments for multiple comparisons? " exact statistical test results, e.g., P values = x but not P values < x; " definition of ‘center values’ as median or average; " definition of error bars as s.d. or s.e.m. 1.a. How was the sample size chosen to ensure adequate power to detect a pre-specified effect size? 1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
      28. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria preestablished?
      29. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe. For animal studies, include a statement about randomization even if no randomization was used. 4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. 4.b. For animal studies, include a statement about blinding even if no blinding was done
      30. For every figure, are statistical tests justified as appropriate? Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Is there an estimate of variation within each group of data? Is the variance similar between the groups that are being statistically compared? Manuscript Number: EMM-2017-08547 EMBO PRESS A- Figures Reporting Checklist For Life Sciences Articles (Rev. June 2017) This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal’s authorship guidelines in preparing your manuscript. PLEASE NOTE THAT THIS CHECKLIST WILL BE PUBLISHED ALONGSIDE YOUR PAPER Journal Submitted to: EMBO molecular medicine Corresponding Author Name: Masahisa Katsuno a statement of how many times the experiment shown was independently replicated in the laboratory. Any descriptions too long for the figure legend should be included in the methods section and/or with the source data. In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects. definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.). Please fill out these boxes # (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name). C- Reagents B- Statistics and general methods the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
      31. Data the data were obtained and processed according to the field’s best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:
      32. Captions The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation. YOU MUST COMPLETE ALL CELLS WITH A PINK BACKGROUND # Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). Based on previous studies, we estimated that a sample size of 20 mice per group would provide 80% power to detect 0.33 hazard-ratio for survival with 30-week observation between treatment groups (Log-Rank test), with a two-sided α level of 0.05 (Page 23). NA We randomly allocated the mice to each treatment group (Page 15). We randomly allocated the mice to each treatment group (Page 15). The investigater blindly performed the behavior tests (Page 15). The investigater blindly performed the behavior tests (Page 15). Yes. We described the statistical methods in each legend. Yes. We confirmed the normality of data. Yes. We tested them using ANOVA. Yes. We confirmed the variance was similar between groups.
      33. To show that antibodies were profiled for use in the system under study (assay and species), provide a citation, catalog number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right).
      34. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination.
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      36. Report species, strain, gender, age of animals and genetic modification status where applicable. Please detail housing and husbandry conditions and the source of animals.
      37. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the committee(s) approving the experiments.
      38. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, 2010) to ensure that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.
      39. Identify the committee(s) approving the study protocol.
      40. Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
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      44. For phase II and III randomized controlled trials, please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission. See author guidelines, under ‘Reporting Guidelines’. Please confirm you have submitted this list.
      45. For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right). See author guidelines, under ‘Reporting Guidelines’. Please confirm you have followed these guidelines. 18: Provide a “Data Availability” section at the end of the Materials & Methods, listing the accession codes for data generated in this study and deposited in a public database (e.g. RNA-Seq data: Gene Expression Omnibus GSE39462, Proteomics data: PRIDE PXD000208 etc.) Please refer to our author guidelines for ‘Data Deposition’. Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Functional genomics data e. Proteomics and molecular interactions
      46. Deposition is strongly recommended for any datasets that are central and integral to the study; please consider the journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right).
      47. Access to human clinical and genomic datasets should be provided with as few restrictions as possible while respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public accesscontrolled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right).
      48. Computational models that are central and integral to a study should be shared without restrictions and provided in a machine-readable form. The relevant accession numbers or links should be provided. When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.
      49. Could your study fall under dual use research restrictions? Please check biosecurity documents (see link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could. F- Data Accessibility D- Animal Models E- Human Subjects We confirmed the guideline. G- Dual use research of concern We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page23 (section of Data Availability in the Material and Method). The acession No. will be added later. We described the information on Page16-17 (section of Immunoblotting, and Histology and immunohistochemistry in the Material and Method). We described the details of cell lines on Page 6,8,9,and 22 (section of Result and Material and Method). We tested mycoplasma contamination. We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page15 (section of Animals in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). We described the information on Page16 (section of Autopsy specimens in the Material and Method). NA NA NA NA NA NA NA NA
    1. This plain page incorporates an overview of job aids by Allison Rossett, who is the foremost authority on the topic. Not all information is given away for free as she wants to sell her books, which are also promoted on the page. This page can be a good way of tracking her current work. Rating 3/5

    1. CourseFolk

      I've taken the focus off CourseFolk being educators who craft the lessons and made that something The Lady Alliance does and CourseFolk just does the techy website/members stuff. I think influencers will balk at a random company doing the lesson writing part for them. I'm still happy to do the interviews and workshop the lessons, but maybe I can do that with a Lady Alliance tag somehow. We can chat more about how this will work

  7. Feb 2019
    1. the agent model swaps the in-put and output sequences, and it also takes the tag of filledinformation slots as an input which is extracted from dia-logue in previous turns by pattern matching with the orderinformation in ground truth

      agent model 构建前先预训练。网络结构和user model一样,但是输入和输出反转,同时也把之前对话中已经填充的槽位信息作为输入。但是这俩部分信息并不是简单的直接拼接在一起,而是来学习适合的attention 权重来更好的利用注意力机制。此外任何其他额外的语义意图标签都不必用。

    1. And who will write the software that makes this contraption useful and productive? We will. In fact, we’re already doing it, each of us, every day. When we post and then tag pictures on the community photo album Flickr, we are teaching the Machine to give names to images. The thickening links between caption and picture form a neural net that can learn. Think of the 100 billion times per day humans click on a Web page as a way of teaching the Machine what we think is important. Each time we forge a link between words, we teach it an idea. Wikipedia encourages its citizen authors to link each fact in an article to a reference citation. Over time, a Wikipedia article becomes totally underlined in blue as ideas are cross-referenced. That massive cross-referencing is how brains think and remember.

      This was the main goal of the 'docuverse' where information will be cross referenced and everything will be related in someway to everything else.

    1. tag those and only those who will really derive benefit

      Implies you know who those people are and you may not know but for some of us that is the whole point of an ESN

    2. Developing employees

      An HBR tag for the article

    1. We're working on an improved

      Do we just post to public or do we add a LDRS 626 tag?

    1. A study published in October of last year sought to determine how to make best use of digital out-of-home (DOOH) advertisements in the London Underground.7 An example of a DOOH ad would be a digital billboard programed to change the advertisement on display after a specific period of time.  To achieve their goal, the researchers used the same Twitter Streaming API described in the previous study; however, this time they utilized Twitter’s geotagging function (a capability that allows Twitter users to “tag” their location when they post a tweet). Each London Underground station was carefully outlined on a map of London.  Then, the researchers randomly sampled geotagged tweets falling within those zones (meaning the tweeter was at a station).  The specific Underground station, the time of the tweet, and the content of the tweet were all extracted. The researchers continued this practice for one year, seemingly unbeknownst to the Twitter-using patrons of the London Underground, collecting over 10.5 million tweets. This data was then compiled and processed to determine what sort of things people were tweeting about in each London Underground station at certain times of the day on weekdays and on weekends.  For example, nearly 35% of tweets from the Holloway Road station were about sports, and almost 40% of tweets posted between 6 PM and midnight on weekends at the North Greenwich station were about music.8 The authors of the study recommended using this data to create targeted DOOH advertising. For instance, a music-related ad on a rotating digital billboard at night on the weekends in North Greenwich station would probably be more successful than an ad for a sports team.

      This entire passage gives a perfect example of how the data can be broken down and used to make money by advertisers. I intend to allude to the fact that it doesn't stop here.

    1. How do you manage information flows? If anyone is using a personal wiki-style long term information tool I’d love to hear from you!

      I've got a handful of interesting things bookmarked here: https://boffosocko.com/tag/wikis/ which includes a rabbit hole of a request similar to your own.

    1. <p>This is a paragraph.</p> <p>This is another paragraph.</p>

      Paragraph Tag

    1. <a href="default.asp">  <img src="smiley.gif" alt="HTML tutorial" style="width:42px;height:42px;border:0;"></a>

      Image as a link - Ignore the style="…." part of the tag. Inline styles are bad!

    2. <img src="https://www.w3schools.com/images/w3schools_green.jpg" alt="W3Schools.com">

      Image Tag

    1. <a href="https://www.w3schools.com/html/">Visit our HTML tutorial</a>

      Link Tag

    1. few

      Is there a common tag that we are using for annotations here?

    1. with needle-sort retrieval

      My dad, who was at the time into educational technology, used to show us that trick. You'd have a pile of punch cards, for which the notches would represent tags. To select the cards matching a tag, you'd stick a needle into the deck, spin it, then page through whatever didn't fall out onto your desk.

    1. it allows you to tag records

      Since DH is such a collaborative subject, you have to make sure references are accurate so that everyone gets credit for their contributions

  8. Jan 2019
    1. . The nofollow tag came about as a method to help stop automated blog comment, guest book, and link injection spam

      Appreciated

    1. SGML, HTML, XML

      SGML = Standard Generalized Markup Language, "a standard for how to specify a document markup language or tag set... SGML is not in itself a document language, but a description of how to specify one." So a set of guidelines, like TEI?

    1. $10 billion is a hefty price tag.

      however Dr. Hossenfelder does not agree the cost outweighs the benefits

    2. With a $5 billion price tag and a $1 billion annual operation cost, the L.H.C. is the most expensive instrument ever built

      That is intense. 5 billion $ to build, yet it cost 1 billion to upkeep.

    1.  .   Social Media is where we will connect with the broader digital storytelling community, which are very active on Twitter and Instagram.  You may participate anonymously if you prefer.  Tag all class related communications with #inte5340 (Links to an external site.)Links to an external site. and #digstory (Links to an external site.)Links to an external site. .

      Would using our personal social media be acceptable for public communication or would you suggest in creating a new account?

    2. Articles and videos will be assigned each week.

      Just wondering what the weekly deadline for discussion will be. Also, do I need to tag these comments...I just saw the option below?

    3. Tag all related posts

      So, we will not be sharing the blog directly on canvas, only tagging our posts correct?

    1. Astros outfielder Josh Reddick is a big wrestling fan, so after he tied the knot this weekend, him and his bride came out to their friends and family using the same entrace as current WWE tag team champions Bobby Roode and Chad Gable.

      "him and his bride came out to their friends"

    1. Machines with interchangeable parts can now be constructed with great economy of effort. In spite of much complexity, they perform reliably. Witness the humble typewriter, or the movie camera, or the automobile. We have reached a point in technology now where all of these inventions can perform their jobs without human involvement.

      His description of the "humble typewriter" seems like such an understatement in comparison to the computers and recording devices we now have

    1. When I received Chris’s comment, my first response was that I should delete my post or at least the incorrect part of it. It’s embarrassing to have your incorrect understandings available for public view. But I decided to leave the post as is but put in a disclaimer so that others would not be misled by my misunderstandings. This experience reminded me that learning makes us vulnerable. Admitting that you don’t know something is hard and being corrected is even harder. Chris was incredibly gentle in his correction. It makes me think about how I respond to my students’ work. Am I as gentle with their work as Chris was to mine? Could I be more gentle? How often have I graded my students’ work and only focused on what they did wrong? Or forgotten that feeling of vulnerability when you don’t know something, when you put your work out for others to judge? This experience has also reminded me that it’s important that we as teachers regularly put ourselves into situations in which we authentically grapple with not knowing something. We should regularly share our less than fully formed understandings with others for feedback. It helps us remember that even confident learners can struggle with being vulnerable. And we need to keep in mind that many of our students are not confident learners.

      I'm reminded here of the broad idea that many bloggers write about sooner or later of their website being a "thought space" or place to contemplate out in the open. More often than not, even if they don't have an audience to interact with, their writings become a way of thinking out loud, clarifying things for themselves, self-evolving, or putting themselves out there for potential public reactions (good, bad, or indifferent).

      While writing things out loud to no audience can be helpful and useful on an individual level, it's often even more helpful to have some sort of productive and constructive feedback. While a handful of likes or positive seeming responses can be useful, I always prefer the ones that make me think more broadly, deeply, or force me to consider other pieces I hadn't envisioned before. To me this is the real value of these open and often very public thought spaces.

      For those interested in the general idea, I've been bookmarking/tagging things around the idea of thought spaces I've read on my own website. Hopefully this collection helps others better understand the spectrum of these ideas for themselves.

      With respect to the vulnerability piece, I'm reminded of an episode of <cite>The Human Current</cite> I listened to a few weeks back. There was an excellent section that touched on building up trust with students or even a class when it comes to providing feedback and criticism. Having a bank of trust makes it easier to give feedback as well as to receive it. Here's a link to the audio portion and a copy of the relevant text.

    1. burying their old professional competition in the demand of a common cause, have shared greatly and learned much. It has been exhilarating to work in effective partnership. Now, for many, this appears to be approaching an end.

      What is interesting is how individuals still share their knowledge to create new things, artists and scientists collaborating together, but how there is still that tight restriction that controls what can be done. New inventions, artworks, music, and tools are still copyrighted and individuals must apply to work with them. You also have inventors that place an outrageous price tag on some of their products, limiting the number of people who can buy them and find out what things they can do that are beyond even the mind of the tool's creator.

    1. Hypothesis Annotation instructions:

      1) Make 2-3 annotations. 2) If another student has already annotated a passage you wished to annotate, you have two options: reply to that annotation or choose another passage to annotate. 3) Reply to at least 2 annotations made by your peers 4) If someone replies to your annotation, reply to keep the conversation going. 5) TAG all annotations CITA2019

    1. New York City Fire Department

      Sample, notice how I made it public and used a tag. You can also add links to other material.

      Images are good too, but only if you think they add something to our understanding of the text. Here's the first female firefighter in the NYFD.

    1. Proche de la basilique Saint Sernin, haut lieu touristique de la ville rose, le quartier Arnaud Bernard ne profite pas de ce rayonnement. Beaucoup de rues sont sales et le quartier n’attire pas les visiteurs. Des tags et des graffs décorent les murs de certains immeubles. Le quartier fait partie des tout premiers de la ville à accueillir de nombreux artistes graffeurs, mais sans organisation. En 1997 l’association « carrefour culturel » décide de lancer un projet avec les habitants du quartier: réaliser une fresque géante dans la rue Gramat pour couvrir les tags. Après trois ans de discussions avec la municipalité, les propriétaires et les artistes, la fresque peut enfin voir le jour. Les riverains participent eux-mêmes au nettoyage de la rue pour accueillir les dessins. Elle est au cœur d’un projet citoyen. David Brunel explique :  » le but c’est de créer à l’époque des actions culturelles où les citoyens sont à l’initiative, mais aussi acteur du projet du début à la fin » Il poursuit :  » le quartier est vu comme une base d’expérience, avec une réflexion sur le civique ». Parmi les artistes, on retrouve un SDF du quartier, un peintre mais aussi des gens venu d’Espagne. Ils sont tous là pour donner une vision de la ville et de son histoire. Plus qu’une fresque ce sont des échanges, un projet construit par les habitants, pour les habitants. Annick Lodereau, l’une des meneuses, résume le projet à l’époque :  » C’est une fresque anti-tag, une création qui démontre que l’on peut faire des choses ensemble et leur donner un sens ». Malgré l’ampleur du projet, au fil des ans la rue est de nouveau utilisée par les tagueurs qui repassent sur les fresques de l’époque dont il ne reste aujourd’hui plus rien.

      Fais des retours ligne pour marque les paragraphes, sinon c'est trop difficile à lire.

    1. It isn't rocket science, but as Jon indicates, it's incredibly powerful.

      I use my personal website with several levels of taxonomy for tagging and categorizing a variety of things for later search and research.

      Much like the example of the Public Radio International producer, I've created what I call a "faux-cast" because I tag everything I listen to online and save it to my website including the appropriate <audio> link to the.mp3 file so that anyone who wants to follow the feed of my listens can have a playlist of all the podcast and internet-related audio I'm listening to.

      A visual version of my "listened to" tags can be found at https://boffosocko.com/kind/listen/ with the RSS feed at https://boffosocko.com/kind/listen/feed/

    1. In this section of the paper we broach two aspects of this articulation issue, onefocusing on the management of workflow, the other on the construction and manage-ment of what we term a ‘common information space’. The former concept has beenthe subject of discussion for some time, in the guise of such terms as office automa-tion and more recently, workflow automation. The latter concept has, in our view,been somewhat neglected, despite its critical importance for the accomplishmentof many distributed work activities

      A quick scan of ACM library papers that tag "articulation work" seems to indicate the "common information space" problem still has not attracted a lot of study. This could be a good entry point for my work with CSCW because time cuts across both workflow and information space.

      Nicely bundles boundary infrastructure, sense-making and distributed work