Dual specificity phosphatase 7 drives the formation of cardiac mesoderm in mouse embryonic stem cells

Stanislava Sladeček; Katarzyna Anna Radaszkiewicz; Martina Bőhmová; Tomáš Gybeľ; Tomasz Witold Radaszkiewicz; Jiří Pacherník

doi:10.1371/journal.pone.0275860

Peer Review History

Original SubmissionApril 5, 2022
7 Jun 2022 Decision Letter - Johnson Rajasingh, Editor PONE-D-22-10089Dual Specificity Phosphatase 7 Drives the Formation of Cardiac Mesoderm in Mouse Embryonic Stem CellsPLOS ONE Dear Dr. Pachernik, 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 revise your manuscript by addressing all the comments raised by the reviewers that are given below. Please submit your revised manuscript by Jul 01 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. 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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: Yes ****** 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 this article, the authors analyzed the role of DUSP7 in cardiac differentiation using mouse embryonic stem cells. They utilized CRISPR technology to generate DUSP7 KO cells and supplemented their results with qPCR and Western Blot analysis. Overall, I recommend this paper be accepted with minor revisions because I believe the abstract and some of the figures need improvement. Additionally, I have a couple of questions/concerns about their cell studies. The abstract should focus on the work done by the authors only and should not include any references. The cells were cultured for more than 40 passages, did they perform any karyotyping to determine if any chromosomal abnormalities were present in the cells after long shelf life? Why not perform overexpression of DUSP7 if you are talking about it in your discussion? I believe overexpression studies would have given strong support to your paper. In Figures 1B and 1D, for example, the font is too small and some of the labels are not legible. The quality of Figure 3 is very poor, either improve the resolution or substitute for the supplemental immunostaining pictures. Figure 5 needs to show a cardiomyocyte-specific marker/antibody. Reviewer #2: Sladecek et al. research article titled “Dual specificity phosphatase 7 drives the formation of cardiac mesoderm in mouse embryonic stem cells” focuses on the role of DUSP7 in mouse embryonic stem cells and elucidates that the DUSP7 is more important for early neural and cardiac mesoderm development by in vitro. The data presented in this study strongly support the conclusion drawn by the authors. Some of the minor concerns are noted below to improve this manuscript before publishing. 1. Typo errors need to be checked. a. In the abstract, there are some numerical numbers in each line (21 -31). It doesn’t make any meaning. b. In section 3.5, DUSP7 is mentioned in small/capital letters. Please make it uniform throughout the manuscript. 2. In fig. 5A, the scale bar was not visible clearly. 3. The resolution of the figures is not good. Importantly the figure.1 text is hard to read. Please provide high quality figures. ****** 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: No 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.0275860.r001
Revision 1
29 Jun 2022 Author Response Dear editor and reviewers, Thank you very much for your comments to our manuscript. Please find bellow our responses to individual raised points. We hope they will be to your satisfaction. Reviewer #1: The abstract should focus on the work done by the authors only and should not include any references. References, numbers from the abstract, have been removed. The cells were cultured for more than 40 passages, did they perform any karyotyping to determine if any chromosomal abnormalities were present in the cells after long shelf life? All experiments in which cells were differentiated as well as experiments based on which we were drawing conclusions on DUSP7’s effect on phosphorylation have been done in cells cultured 5-20 passages. The high passage was only used in experiment involving markers of pluripotency (Fig. 2D) to see if there will be some change in our KO cell lines in pluripotency even after longer shelf life. However, since we did not see any difference between the KO and WT cells, we did not investigate further changes that this kind of long-term cultivation could have. We have not performer karyotyping of the cells ourselves, but the used mouse embryonic stem cells line R1 (from which also all the KO cells lines were derived) is well established and defined cell line, which we have bought from ATCC (American Tissue Culture Collection). Why not perform overexpression of DUSP7 if you are talking about it in your discussion? I believe overexpression studies would have given strong support to your paper. Currently we are still working on determining the importance of DUSP7 as well as couple of other DUSPs in the development of early mesoderm and cardiomyocytes, where our preliminary results point to their importance in different stages of development. In that context, we agree that it would be very interesting to investigate the overexpression of DUSP7. However, this would be more interesting when it comes to pinpointing the importance of DUSPs in different stages, developmental intervals, than overall general effect that DUSP7 has in in vitro development from mouse ES cells, which we report in this paper. In Figures 1B and 1D, for example, the font is too small and some of the labels are not legible. We have exported pictures in high resolution and hope that this solves the issue of the labels not being legible. The quality of Figure 3 is very poor, either improve the resolution or substitute for the supplemental immunostaining pictures. We have exported the figures in high resolution (330dpi) which we hope resolves the issue. The pictures of embryoid bodies in Figure 3 were taken in hanging drops on stereomicroscope which does not provide very good quality of magnification. Furthermore, embryoid bodies are floating 3D objects that are difficult to focus on. Their pictures were used here more as an illustration to the graph next to them which shows the size of embryoid bodies on day 5. We cannot substitute them with immunostaining pictures from supplements, since those are taken on day 20 and have been cultured as adherent cells for 15 days. We hope that the quality in which they are now exported is acceptable. Figure 5 needs to show a cardiomyocyte-specific marker/antibody. We have changed the description of this figure, which now states that MF20 was used antibody instead of MHC. As stated in the material and methods (table 2) we used clone MF20 from the Developmental Studies Hybridoma Bank, which is specific for myosin heavy chain 6 and 7, which are considered as cardiomyocyte-specific markers. Reviewer #2: Typo errors need to be checked. a. In the abstract, there are some numerical numbers in each line (21 -31). It doesn’t make any meaning. This has been addressed and the numbers have been erased b. In section 3.5, DUSP7 is mentioned in small/capital letters. Please make it uniform throughout the manuscript. We call our cell lines DUSP7 KOa/b/c and generally when speaking about DUSP7, to have it more uniformed, we treat it as speaking about the mouse protein rather than gene and therefore write it all in capital letters. However, in case of section 3.5 as well as when its mentioned in Table 1 we are explicitly talking about measuring the gene expression of DUSP7 and therefore we used the the nomenclature which is used for mouse genes – italicized, with only the first letter in upper-case. We had mistaken in this regard in section 2.5 and Figure 1 which has been corrected. In fig. 5A, the scale bar was not visible clearly. We modified scale bar in fig. 5A and added it (Scale bar represents 100um) into the description of fig.5. The resolution of the figures is not good. Importantly the figure.1 text is hard to read. Please provide high quality figures. We have exported figures in high quality (330 dpi) which we hope resolves the issue. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0275860.r002
26 Sep 2022 Decision Letter - Johnson Rajasingh, Editor Dual Specificity Phosphatase 7 Drives the Formation of Cardiac Mesoderm in Mouse Embryonic Stem Cells PONE-D-22-10089R1 Dear Dr. Pachernik, 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, Johnson Rajasingh, Ph.D, HCLD 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: All comments have been addressed 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: The authors addressed all the reviewer's comments and corrected the figures specified in the first revision. Reviewer #2: The Authors have rectified and addressed all the pointed comments in the revised version of the manuscript. ****** 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 ******** https://doi.org/10.1371/journal.pone.0275860.r003
Formally Accepted
30 Sep 2022 Acceptance Letter - Johnson Rajasingh, Editor PONE-D-22-10089R1 Dual specificity phosphatase 7 drives the formation of cardiac mesoderm in mouse embryonic stem cells Dear Dr. Pacherník: 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. Johnson Rajasingh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0275860.r004

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