PONE-D-23-40786RIO-kinase 2 is essential for hematopoiesisPLOS ONE

Dear Dr. Helin,

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 Feb 22 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.

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,

Jimin Han

Academic Editor

PLOS ONE

Journal requirements:

1. When submitting your revision, we need you to address these additional requirements.

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. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

3. Thank you for stating the following in the Competing Interests section: 

[K.H. is a consultant for Dania Therapeutics Aps and a scientific advisor for Hannibal Health Innovation. The other authors declare no competing interests.]. 

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: ""This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. 

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

4. 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.

5. We note that Figure 1A, 1E, 2A and 2E in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1A, 1E, 2A and 2E to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an ""Other"" file with your submission. 

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

Additional Editor Comments:

Dear Dr. Kristian Helin

This research paper, titled " RIO-kinase 2 is essential for hematopoiesis" explores the role of RIO-kinase 2 (RIOK2) in hematopoiesis, the process of blood cell formation. The study uses conditional knockout mouse models to demonstrate that RIOK2 is crucial for the differentiation and maintenance of hematopoietic stem and progenitor cells, as well as for the survival of fully differentiated blood cells. The absence of RIOK2 leads to rapid death in mice, indicating its vital role in the hematopoietic system. This research provides insights into the potential targeting of RIOK2 for treating blood cell-related disorders. The whole story is quite in-depth and it’s very interesting. I'm including feedback from two expert reviewers regarding your manuscript. Their insights highlight the study's potential while also presenting various critiques and recommendations. After read reviewer’s feedback, I believe this article requires careful revision. So, I decide give you major revision decision.

It's our hope that you find this feedback constructive. Should additional data be available to address the raised concerns, we are open to examining a modified submission. Our timeline is adaptable for any necessary extended research. For submissions linked to a specific thematic issue, please liaise with the editorial team concerning resubmission deadlines. We emphasize that key findings must be backed by comprehensive statistics from ample independent trials. Manuscripts may be returned at the editor's discretion for the completion of any lacking or partial statistical data.

I strongly suggest rearranging the figures, so they are more reader-friendly and more clearly. For more details, please refer to the layout instructions of PLOS ONE.

Yours sincerely,

Jimin Han

Academic Editor,

PLOS ONE

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 study explores the role of RIOK2 in blood cell formation, both in vivo and in vitro, with the manuscript conveying an evident phenotype associated with the knockout of RIOK2.

Major Point:

The figures included in the main text are of such low quality that it is difficult to discern the text within the images. Enhancement of image clarity is necessary.

Minor Points:

1. In Fig S1C, concerning the lower image of genotyping, it is unclear what the genotype is for the control. Why do two lanes of ctrl display mismatched bands?

2. Is it critical to establish the baseline levels of CD45.1 and CD45.2 in mice before the transplantation process? Additionally, would it enhance clarity to more distinctly show the reduction of CD45+ cells following radiation?

3. Some of the statistical charts, like those in Figure 1B, 1C, and FigS1D, would benefit from an analysis of significance to support the findings.

Reviewer 2:

RIOK2 is important for hematopoietic stem cell（HPSC）maintance and differentiation. This manuscript investigates the effects of RIOK2 on HPSC function and HPSC transplantation using a conditional knockout mouse model in the hematopoietic system. The research findings have significant implications for understanding HPSC function and transplantation. However, revisions need to be made to the manuscript before it can be accepted.

In Figure 1, we propose that both Riok2fl/fl; Rosa26::CreERT2 and Riok2fl/+; Rosa26::CreERT2 have the effect of downregulating RIOK2 gene expression. Why was Riok2+/+; Rosa26::CreERT2 mice not used as a control?

In Supplementary Figure 1, should there be additional images demonstrating the efficiency of RIOK2 gene knockdown in Riok2fl/fl; Rosa26::CreERT2 and Riok2fl/+; Rosa26::CreERT2 mice, rather than solely relying on DNA-level identification?

In Figure 2, the experimental data regarding the impact of Riok2 knockout on HSC proliferation and differentiation is insufficient to support the authors' conclusions. Are there additional evidence available to substantiate these claims?

[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: 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: 

RIOK2 is important for hematopoietic stem cell（HPSC）maintance and differentiation. This manuscript investigates the effects of RIOK2 on HPSC function and HPSC transplantation using a conditional knockout mouse model in the hematopoietic system. The research findings have significant implications for understanding HPSC function and transplantation. However, revisions need to be made to the manuscript before it can be accepted.

In Figure 1, we propose that both Riok2fl/fl; Rosa26::CreERT2 and Riok2fl/+; Rosa26::CreERT2 have the effect of downregulating RIOK2 gene expression. Why was Riok2+/+; Rosa26::CreERT2 mice not used as a control?

In Supplementary Figure 1, should there be additional images demonstrating the efficiency of RIOK2 gene knockdown in Riok2fl/fl; Rosa26::CreERT2 and Riok2fl/+; Rosa26::CreERT2 mice, rather than solely relying on DNA-level identification?

In Figure 2, the experimental data regarding the impact of Riok2 knockout on HSC proliferation and differentiation is insufficient to support the authors' conclusions. Are there additional evidence available to substantiate these claims?

Reviewer #2: The study explores the role of RIOK2 in blood cell formation, both in vivo and in vitro, with the manuscript conveying an evident phenotype associated with the knockout of RIOK2.

Major Point:

The figures included in the main text are of such low quality that it is difficult to discern the text within the images. Enhancement of image clarity is necessary.

Minor Points:

1. In Fig S1C, concerning the lower image of genotyping, it is unclear what the genotype is for the control. Why do two lanes of ctrl display mismatched bands?

2. Is it critical to establish the baseline levels of CD45.1 and CD45.2 in mice before the transplantation process? Additionally, would it enhance clarity to more distinctly show the reduction of CD45+ cells following radiation?

3. Some of the statistical charts, like those in Figure 1B, 1C, and FigS1D, would benefit from an analysis of significance to support the findings.

**********

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: Jia He

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.

RIO-kinase 2 is essential for hematopoiesis

PONE-D-23-40786R1

Dear Dr. Helin,

This time the authors have corrected it in such detail that I think PLOS ONE is ready to accept the paper.I asked the Reviewer to review it, and none of them had any opinion.I firmly believe that this article will contribute to the advancement of this field.

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,

Jimin Han

Academic Editor

PLOS ONE

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 #3: 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 #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: 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 #3: 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 #3: 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 #3: (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 #3: No

**********