The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB

Mai Tuyet Nguyen; Min-Ah Lee; Young-Kook Kim; Hyun Kook; Dongtak Jeong; Seung Pil Jang; Tae Hwan Kwak; Woo Jin Park

doi:10.1371/journal.pone.0269735

Peer Review History

Original SubmissionAugust 11, 2021
6 Oct 2021 Decision Letter - Dhyan Chandra, Editor PONE-D-21-25986The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkBPLOS ONE Dear Dr. Park, 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 during the review process. Please submit your revised manuscript by Nov 20 2021 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Dhyan Chandra, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following financial disclosure: “W.J.P. was supported by grants from National Research Foundation of Korea (2019R1A2C2085457, 2019R1A4A1028534) funded by the Korean Government (MSIP). Y.-K.K. and H. K. were supported by grants from National Research Foundation of Korea (2018R1A2B3001503, 2018R1A2B6001104, 2019R1A4A1028534, 2020R1C1C1006507). D.J. was supported by grants from National Research Foundation of Korea (2021R1A2C1008058, 2021R1A4A5032463)” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: “W.J.P. was supported by grants from National Research Foundation of Korea (2019R1A2C2085457, 2019R1A4A1028534) funded by the Korean Government (MSIP). Y.- K.K. and H. K. were supported by grants from National Research Foundation of Korea (2018R1A2B3001503, 2018R1A2B6001104, 2019R1A4A1028534, 2020R1C1C1006507). D.J. was supported by grants from National Research Foundation of Korea (2021R1A2C1008058, 2021R1A4A5032463)” We note that you have provided funding information within the Acknowledgements Section. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “W.J.P. was supported by grants from National Research Foundation of Korea (2019R1A2C2085457, 2019R1A4A1028534) funded by the Korean Government (MSIP). Y.-K.K. and H. K. were supported by grants from National Research Foundation of Korea (2018R1A2B3001503, 2018R1A2B6001104, 2019R1A4A1028534, 2020R1C1C1006507). D.J. was supported by grants from National Research Foundation of Korea (2021R1A2C1008058, 2021R1A4A5032463)” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 7. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? 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 Reviewer #2: 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: In the submitted manuscript authors evaluated the mechanism of CCN5 mediated apoptosis of myofibroblasts involving p53 and NFkB. Further, SMAD7 was identified as the downstream target of CCN5 using luciferase reporter construct and reported to be positively regulated by CCN5 at transcriptional level. The manuscript is written with clarity and includes sufficient detail in each section. The experimental design is logical and most of the experiments were conducted in satisfactory manner. There are few minor things that authors should address: 1. In figure 1a loading control (GAPDH) seems to be high in FB+TGFβ samples. Authors are encouraged to quantify the western blots by densitometry to confirm if there is any overexpression of P53 and p-p53 (both S392 and S15). 2. Authors could have used high magnification images for Tunnel assay. 3. The statement in the last paragraph of the introduction “cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-�” is little confusing. It seems cardiac MyoFBs were transdifferentiated, though cardiac FBs were differentiated. Authors should review the statement. Reviewer #2: The Manuscript "The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkB" by Nguyen et al is the extension of their earlier work where they have shown that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, authors have the elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. Authors have demonstrated that the role of CCN5 in MyoFB-specific apoptosis using cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-β. They found that a balance between pro-apoptotic p53 and anti-apoptotic NFkB is established in MyoFBs and that CCN5 tips over the balance to favor p53, which results in apoptosis through SMAD7-mediated inhibition of NFkB. In the end, the authors have claimed that this work provides insight into the role of CCN5 in reversing CF and a basis for the development of anti-fibrotic therapies. There are several concerns that need to be addressed. General comments: 1. In the Abbreviations section, "E" of extracellular should be written in the capital later (line-47) 2. Extend the abbreviations "CCN5-5HA" (line- 114) 3. Animal Ethics Statement- should be written properly. It is written very haphazardly Specific comments: 1. In the result section, as the authors mention in the materials and methods, they used GAPDH to validate the expression level of target proteins but they did not normalize the target protein with GAPDH (All western blot figures except fig.5) 2. The authors did not mention the molecular weight of any proteins (All figures) 3. In the result section, the authors use two type samples (FB and FB+ TGFβ2) but the western blot result showed the three bands of each group. If all three bands indicate the same group, then please mention it properly. 4. In Figure1A, the authors mention TGFβ2 but in Figure Legends and result section they mention only TGF-β. Please correct this. 5. What is the rationale of 30mM p53 siRNA dose selection because the authors did not explain the selection? 6. Again, the authors used 10ng/ml TGFβ and 100 nM AngII to transdifferentiate FB to MyoFBs but they did not explain why they used this concentration? 7. Similarly, they did not explain the dose selection of NFkB inhibitor (1µM)? 8. In the result section, sudden introduce that they used profibrotic proteins (TGFβ and AngII) to induced FB into MyoFB but they should be included in material and methods also. 9. In line-216, the authors should use words significantly blocked when the cells were pre-incubated with the p53 siRNA 10. Line-245, the authors used siRNA of SMAD7 but they did not mention the concentration, however, they mentioned the concentration (25mM SMAD7 siRNA) in the representative figure-4A ****** 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? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Ashish Kumar Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0269735.r001
Revision 1
17 Nov 2021 Author Response 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: In the submitted manuscript authors evaluated the mechanism of CCN5 mediated apoptosis of myofibroblasts involving p53 and NFkB. Further, SMAD7 was identified as the downstream target of CCN5 using luciferase reporter construct and reported to be positively regulated by CCN5 at transcriptional level. The manuscript is written with clarity and includes sufficient detail in each section. The experimental design is logical and most of the experiments were conducted in satisfactory manner. There are few minor things that authors should address: 1. In figure 1a loading control (GAPDH) seems to be high in FB+TGFβ samples. Authors are encouraged to quantify the western blots by densitometry to confirm if there is any overexpression of P53 and p-p53 (both S392 and S15). --> We have now quantitative graphs in figures 1~4. 2. Authors could have used high magnification images for Tunnel assay. --> We have now higher mag images with scale bars. 3. The statement in the last paragraph of the introduction “cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-�” is little confusing. It seems cardiac MyoFBs were transdifferentiated, though cardiac FBs were differentiated. Authors should review the statement. --> We revised the sentence to read " In this study, we attempted to further elucidate the role of CCN5 in MyoFB-specific apoptosis. FBs were freshly isolated from rat hearts, and MyoFBs were obtained by trans-differentiating FBs by treatment with TGF-�." Reviewer #2: The Manuscript "The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkB" by Nguyen et al is the extension of their earlier work where they have shown that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, authors have the elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. Authors have demonstrated that the role of CCN5 in MyoFB-specific apoptosis using cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-β. They found that a balance between pro-apoptotic p53 and anti-apoptotic NFkB is established in MyoFBs and that CCN5 tips over the balance to favor p53, which results in apoptosis through SMAD7-mediated inhibition of NFkB. In the end, the authors have claimed that this work provides insight into the role of CCN5 in reversing CF and a basis for the development of anti-fibrotic therapies. There are several concerns that need to be addressed. General comments: 1. In the Abbreviations section, "E" of extracellular should be written in the capital later (line-47) --> Corrected. 2. Extend the abbreviations "CCN5-5HA" (line- 114) --> We inserted a sentence that explains CCN5-HA in line 117~118, which read " We synthesized a gene encoding CCN5 fused with a HA tag at its carboxy terminus and referred to it as CCN5-HA." 3. Animal Ethics Statement- should be written properly. It is written very haphazardly --> We inserted a sentence in lines 93~95, which read " The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996)." Specific comments: 1. In the result section, as the authors mention in the materials and methods, they used GAPDH to validate the expression level of target proteins but they did not normalize the target protein with GAPDH (All western blot figures except fig.5) --> We have now quantitative graphs in figures 1~4. 2. The authors did not mention the molecular weight of any proteins (All figures) --> All western blots now have molecular sizes marked. 3. In the result section, the authors use two type samples (FB and FB+ TGFβ2) but the western blot result showed the three bands of each group. If all three bands indicate the same group, then please mention it properly. --> For two groups (FB, FB+ TGFβ2), we had three independent cultures. Each lane in western blots represent samples of these three independent cultures. We added a sentence, " For each group, three independent cultures were prepared and treated (n=3)." at the ends of each figure legends. 4. In Figure1A, the authors mention TGFβ2 but in Figure Legends and result section they mention only TGF-β. Please correct this. --> In Figure 1A, TGF-β2 is now chaged to TGF-β. 5. What is the rationale of 30mM p53 siRNA dose selection because the authors did not explain the selection? 6. Again, the authors used 10ng/ml TGFβ and 100 nM AngII to transdifferentiate FB to MyoFBs but they did not explain why they used this concentration? 7. Similarly, they did not explain the dose selection of NFkB inhibitor (1µM)? 10. Line-245, the authors used siRNA of SMAD7 but they did not mention the concentration, however, they mentioned the concentration (25mM SMAD7 siRNA) in the representative figure-4A. --> For the issues 5,6,7,10, we now indicate the concentration and more detailed procedure in materials and methods section as well as figure legends. The indicated concentrations were those that were recommended by manufacturors or the best ones when tested in our lab. 8. In the result section, sudden introduce that they used profibrotic proteins (TGFβ and AngII) to induced FB into MyoFB but they should be included in material and methods also. --> This is now included in material and methods section. 9. In line-216, the authors should use words significantly blocked when the cells were pre-incubated with the p53 siRNA --> We added a word "significantly" in line 219. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0269735.r002
5 Apr 2022 Decision Letter - Dhyan Chandra, Editor PONE-D-21-25986R1The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkBPLOS ONE Dear Dr. Park, 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 during the review process. Please submit your revised manuscript by May 20 2022 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Dhyan Chandra, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0269735.r003
Revision 2
29 Apr 2022 Author Response Reviewer #1: In the submitted manuscript authors evaluated the mechanism of CCN5 mediated apoptosis of myofibroblasts involving p53 and NFκB. Further, SMAD7 was identified as the downstream target of CCN5 using luciferase reporter construct and reported to be positively regulated by CCN5 at transcriptional level. The manuscript is written with clarity and includes sufficient detail in each section. The experimental design is logical and most of the experiments were conducted in satisfactory manner. There are few minor things that authors should address: 1. In figure 1a loading control (GAPDH) seems to be high in FB+TGFβ samples. Authors are encouraged to quantify the western blots by densitometry to confirm if there is any overexpression of P53 and p-p53 (both S392 and S15). --> We have now quantitative graphs in figures 1~4. 2. Authors could have used high magnification images for Tunnel assay. --> We have now higher mag images with scale bars. 3. The statement in the last paragraph of the introduction “cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-β” is little confusing. It seems cardiac MyoFBs were transdifferentiated, though cardiac FBs were differentiated. Authors should review the statement. --> We revised the sentence to read " In this study, we attempted to further elucidate the role of CCN5 in MyoFB-specific apoptosis. FBs were freshly isolated from rat hearts, and MyoFBs were obtained by trans-differentiating FBs by treatment with TGF-β." Reviewer #2: The Manuscript "The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB" by Nguyen et al is the extension of their earlier work where they have shown that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, authors have the elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. Authors have demonstrated that the role of CCN5 in MyoFB-specific apoptosis using cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-β. They found that a balance between pro-apoptotic p53 and anti-apoptotic NFκB is established in MyoFBs and that CCN5 tips over the balance to favor p53, which results in apoptosis through SMAD7-mediated inhibition of NFκB. In the end, the authors have claimed that this work provides insight into the role of CCN5 in reversing CF and a basis for the development of anti-fibrotic therapies. There are several concerns that need to be addressed. General comments: 1. In the Abbreviations section, "E" of extracellular should be written in the capital later (line-47) --> Corrected. (Line 50) 2. Extend the abbreviations "CCN5-5HA" (line- 114) --> We inserted a sentence that explains CCN5-HA in line 121~122, which read " We synthesized a gene encoding CCN5 fused with a HA tag at its carboxy terminus and referred to it as CCN5-HA." 3. Animal Ethics Statement- should be written properly. It is written very haphazardly --> We inserted a sentence in lines 93~95, which read " The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996)." Specific comments: 1. In the result section, as the authors mention in the materials and methods, they used GAPDH to validate the expression level of target proteins but they did not normalize the target protein with GAPDH (All western blot figures except fig.5) --> We have now quantitative graphs in figures 1~4. 2. The authors did not mention the molecular weight of any proteins (All figures) --> All western blots now have molecular sizes marked. 3. In the result section, the authors use two type samples (FB and FB+ TGFβ2) but the western blot result showed the three bands of each group. If all three bands indicate the same group, then please mention it properly. --> For two groups (FB, FB+ TGFβ2), we had three independent cultures. Each lane in western blots represent samples of these three independent cultures. We added a sentence, " For each group, three independent cultures were prepared and treated (n=3)." at the ends of each figure legends. 4. In Figure1A, the authors mention TGFβ2 but in Figure Legends and result section they mention only TGF-β. Please correct this. --> In Figure 1A, TGF-β2 is now changed to TGF-β. 5. What is the rationale of 30mM p53 siRNA dose selection because the authors did not explain the selection? 6. Again, the authors used 10ng/ml TGFβ and 100 nM AngII to transdifferentiate FB to MyoFBs but they did not explain why they used this concentration? 7. Similarly, they did not explain the dose selection of NFkB inhibitor (1µM)? 10. Line-245, the authors used siRNA of SMAD7 but they did not mention the concentration, however, they mentioned the concentration (25mM SMAD7 siRNA) in the representative figure-4A. --> For the issues 5,6,7,10, we now indicate the concentration and more detailed procedure in materials and methods section as well as figure legends. The indicated concentrations were those that were recommended by manufacturers or the best ones when tested in our lab. 8. In the result section, sudden introduce that they used profibrotic proteins (TGFβ and AngII) to induced FB into MyoFB but they should be included in material and methods also. --> This is now included in material and methods section. 9. In line-216, the authors should use words significantly blocked when the cells were pre-incubated with the p53 siRNA --> We added a word "significantly" in line 233. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0269735.r004
27 May 2022 Decision Letter - Dhyan Chandra, Editor The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkB PONE-D-21-25986R2 Dear Dr. Park, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Dhyan Chandra, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? 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 Reviewer #2: Yes ****** 6. 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: (No Response) Reviewer #2: (No Response) ****** 7. 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?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Attachments Attachment Submitted filename: PONE_comments.docx https://doi.org/10.1371/journal.pone.0269735.r005
Formally Accepted
22 Jul 2022 Acceptance Letter - Dhyan Chandra, Editor PONE-D-21-25986R2 The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkB Dear Dr. Park: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Dhyan Chandra Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0269735.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .