Transcriptional control of a stem cell factor nucleostemin in liver regeneration and aging

Xiaoqin Liu; Junying Wang; Fang Li; Nikolai Timchenko; Robert Y. L. Tsai

doi:10.1371/journal.pone.0310219

Peer Review History

Original SubmissionMay 23, 2024
16 Jun 2024 Decision Letter - Matias A Avila, Editor PONE-D-24-19975Transcriptional Control of A Stem Cell Factor Nucleostemin in Liver Regeneration and AgingPLOS ONE Dear Dr. Tsai, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised by the reviewer. Please submit your revised manuscript by Jul 31 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This article aims to elucidate the transcriptional mechanisms of nucleostemin (NS) expression during liver regeneration during both liver regeneration in aged livers. The study shows that C/EBPα binding to the NS promoter suppresses its transcriptional activity, leading to reduced NS in this context. In order for this manuscript to be accepted, it is necessary to conduct additional experiments and/or determinations, and clarify certain aspects of the results and discussion, along with correcting certain formal aspects 1. Sham-operated animals should be included to analyze whether the variation in NS levels could be due to the acute-phase response of the laparotomy. 2. Did you observe a delay in the liver index in elderly mice at the end of the experiment? These data should be shown or discussed. 3. What is the significance of NS increase in liver regeneration? You should provide any determination of the impact of defective NS regulation on liver regeneration in elderly mice. 4. It is known that C/EBPα levels change with the circadian rhythm. How could this oscillation affect NS expression? The nutritional status of the mice during the experiment should be explained in the methods. 5. Although basal NS mRNA levels are lower in adult and aged livers, the elevation following partial hepatectomy resembles that of young livers. This suggests that the regulatory mechanisms are similar. It is essential to analyze NS protein levels at selected time points after partial hepatectomy (PH). Do NS and C/EBP (inversely) correlate when performing Pearson correlation analysis? 6. Although EMSA has already demonstrated that C/EBPα binds to the consensus sequences in the NS promoter in vitro, conducting a ChIP would allow verification of this interaction within the context of a chromosome in living cells. 7. Why do AAVs have no effect on young mice? Why did you use 24-month-old mice in AAV studies? Why do you perform a 30% hepatectomy and not a 70% hepatectomy? You need to hypothesize why AAV does not work in young mice but does in old mice. 8. The statistical methods are not described. Formal aspects: 1. The title "aging livers" in Figure 2 is inappropriate. In other graphs, the titles do not correctly inform what is being represented. 2. Graphs in Figure 5 would be clearer if the gene titles were included and the basal data were combined with the hepatectomized liver data in the same graph. 3. Graphs 6B1 and 6C1 would be better understood as line graphs (time series). 4. In some graphs, the curved lines indicating significance are not used correctly. Review and standardize their use. 5. Please correct "C/ebp alpha" written in lowercase. Standardize its writing. 6. Some points are missing (at the end of sentences). ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? 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Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0310219.r001
Revision 1
16 Aug 2024 Author Response We sincerely thank the academic editor and the reviewer for their valid and constructive comments on our manuscript from many angles. We have addressed all the raised critiques by providing new data (Figs. 1A3, 1B, 4B, and S1), new analyses, better clarifications, and justifications (all marked in red in the manuscript). Our point-to-point responses are listed as follows. Academic editor 1. Make sure our manuscript meets PLOS ONE's style requirements: checked 2. In the Methods section, provide additional information regarding the experiments involving animals and ensure you have included details on methods of sacrifice: provided 3. Please include this amended Role of Funder statement in your cover letter: included 4. expand the acronym “NIA-PHS”: done 5. Update your Funding Statement: The funding statement of two funders (NIA-PHS and CPRIT) in the manuscript is correct. 6. Complete Data Availability Statement in the submission form: A Data Availability Statement is provided in the manuscript. 7. Upload original uncropped and unadjusted images: EMSA images were included in the Supplemental Information (Fig. S2). Reviewer 1. Sham-operated animals should be included to analyze whether the variation in NS levels could be due to the acute-phase response of the laparotomy. Response: We have performed sham-operation on 2-month-old mice to determine the NS response to the laparotomy without PHx. The result, now shown in Fig. 1A3, demonstrates that the upregulation of NS is not caused by a response to laparotomy. NS increase during liver regeneration is consistent with previous findings (PMID: 22775537 and 23813570). 2. Did you observe a delay in the liver index in elderly mice at the end of the experiment? These data should be shown or discussed. Response: Liver-to-Body Weight Ratios at 1, 2, 4, and 7 days after 70% PHx at 2 and 16 m/o are presented in Fig. S1. 3. What is the significance of NS increase in liver regeneration? You should provide any determination of the impact of defective NS regulation on liver regeneration in elderly mice. Response: Determination of the impact of defective NS regulation on liver regeneration is interesting but beyond the scope of this study. The focus of this study is to investigate the transcriptional control of NS. The impact of NS loss on liver regeneration was examined using an Albumin-Cre driven NS-knockout mouse model in CCl4-induced liver regeneration (PMID: 23813570), which was cited in the Introduction. 4. It is known that C/EBPα levels change with the circadian rhythm. How could this oscillation affect NS expression? The nutritional status of the mice during the experiment should be explained in the methods. Response: While the effect of many environmental factors (e.g., circadian rhythm, nutrition, etc) in regulating NS expression and hence affecting liver regeneration and aging is potentially interesting but beyond the scope of this study. We appreciate the comment and include this idea in the conclusion statement. We also include the following statement in the Method section: Mice were housed in a vivarium with the light-dark cycle set at 12h-12h with lights on at 06:00 and off at 18:00, and allowed free access to normal chow diet and water. 5. Although basal NS mRNA levels are lower in adult and aged livers, the elevation following partial hepatectomy resembles that of young livers. This suggests that the regulatory mechanisms are similar. It is essential to analyze NS protein levels at selected time points after partial hepatectomy (PH). Do NS and C/EBP (inversely) correlate when performing Pearson correlation analysis? Response: Anti-NS IHC results at selected time points after PHx were provided in Fig. 1B. Pearson correlation analysis showed that the baseline expression of NS and C/EBPα were inversely correlated in young (2-m/o) and old (16-m/o) livers (r= -0.42, p = 0.052), and the regulated expression of NS and C/EBPα during liver regeneration (D0, D1, D2, D4, and D7) were also inversely correlated at 2 m/o (r= -0.44, p = 0.001) and 16 m/o (r= -0.30, p = 0.038). 6. Although EMSA has already demonstrated that C/EBPα binds to the consensus sequences in the NS promoter in vitro, conducting a ChIP would allow verification of this interaction within the context of a chromosome in living cells. Response: ChIP assays were performed in living Hepa1-6 cells (mouse HCC cells) to confirm the binding of FLAG-tagged mouse C/EBPα protein to the endogenous NS promoter. The results are presented in Fig. 4B. We used FLAG-tagged mouse C/EBPα for ChIP because we were unable to ChIP the endogenous C/EBPα with the anti-C/EBPα antibody even on the DGAT2 promoter (a positive control). 7. Why do AAVs have no effect on young mice? Why did you use 24-month-old mice in AAV studies? Why do you perform a 30% hepatectomy and not a 70% hepatectomy? You need to hypothesize why AAV does not work in young mice but does in old mice. Response: Young mice have a higher regenerative capability and more NS expression in their livers compared to aged mice. In conjunction with the effect of C/EBP� knockdown on NS expression in aged mice but not in young mice, we hypothesize that the C/EBP�-mediated inhibition on NS transcription occurs mainly at the old age and may be controlled by post-translational modification such as phosphorylation at S193 (included in Discussion). We perform 30% instead of 70% PHx in 24-month mice because the mortality rate of 70% PHx is much higher than 30% PHx at the old age (included in Methods). 8. The statistical methods are not described. Response: Statistical methods were added to the Methods as well as in the Figure legends. 9. The title "aging livers" in Figure 2 is inappropriate. In other graphs, the titles do not correctly inform what is being represented. Response: Titles of “aging livers” were revised to “baseline livers”, although our original usage of the term “aging livers” is to refer to livers in the aging (ongoing) process from 2-to-8-to-16 m/o, not “aged” livers. 10. Graphs in Figure 5 would be clearer if the gene titles were included and the basal data were combined with the hepatectomized liver data in the same graph. Response: Gene symbols were included in the titles. We appreciate the reviewer’s suggestion of combining basal w/ PHx data, but since these two sets of studies were not performed in parallel, we think it is prudent not to combine them. 11. Graphs 6B1 and 6C1 would be better understood as line graphs (time series). Response: Line graphs for 6B1 and 6C1 are provided in Fig. S3. 12. In some graphs, the curved lines indicating significance are not used correctly. Review and standardize their use. Response: non-significant p values were provided for all curved lines. 13. Please correct "C/ebp alpha" written in lowercase. Standardize its writing. Response: C/EBP was used throughout the ms based on the suggestion. In the previous version, we used the lower case to refer to mouse genes and the upper case to refer to human genes. 14. Some points are missing (at the end of sentences). Response: Assuming point means period, we’ve added periods to all the figure legend titles. Please let us know if we miss any additional ones. Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0310219.r002
27 Aug 2024 Decision Letter - Matias A Avila, Editor Transcriptional Control of A Stem Cell Factor Nucleostemin in Liver Regeneration and Aging PONE-D-24-19975R1 Dear Dr. Tsai, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Matias A Avila, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0310219.r003
Formally Accepted
3 Sep 2024 Acceptance Letter - Matias A Avila, Editor PONE-D-24-19975R1 PLOS ONE Dear Dr. Tsai, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Matias A Avila Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0310219.r004

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