PONE-D-22-04392Quantitative Analysis of Weibel-Palade BodiesPLOS ONE

Dear Dr. Liu,

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 all the points raised during the review process.

 Two experts have evaluated the manuscript and agreed that further amanements are needed including the following points:- a clear hypothesis and justification of the study is needed, especially, that WBP density in cutlured endothelial cells is not t new idea and topic. What does the study add to the field in terms of new knwoledge?- Please clearly indicate the number of biological paralllels as well as the number of independent experiments.- add individual data points to figures (in addition to the mean)- junctional staining should be also performed to clearly show cell morphology

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We look forward to receiving your revised manuscript.

Kind regards,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

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(This work was supported by grants/support from the National Hemophilia Foundation (CJN), the Health Resources & Services Administration – Maternal & Child Health (5 H30 MC00008-20-00 to CJN), the National Institutes of Health (P01 HL144457 to CJN)

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

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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

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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: Lui et al have investigated how endothelial cells reaching confluence determines their WPB load. Using HUVECs and ECFCs they find that endothelial cells, when given the opportunity to form confluent monolayers will reach some sort of a plateau in terms of WPB numbers per cell. This confirms data from Howell et al from >15 years ago. Furthermore, they show that the route taken to in vitro confluence, be it higher initial seeding density or prolonged incubation time, has hardly any influence on the WPB plateau. This paper lacks a clear, biologically relevant hypothesis or concept to put the results of these experiments into context or warrant their publication. As such, the value of this paper is very limited.

Minor comments:

Figure 1: The images shown are very grainy. The typical elongated morphology of WPBs is difficult to discern and large clusters of granules stacked on top of on another appear to present. With this kind of detail I am not sure how accurate the quantifications are. Is this a PDF conversion issue?

Reviewer #2: The article describes the effect of confluency on WPB numbers in endothelial cells (both HUVEC and ECFCs). The authors compare WPB number in cells seeded at differing densities and cells allowed to reach confluency over a differing period of time.

The effect of confluency on WPB number has been known for a while but there have been contradictory publications which have somewhat muddied the water and this manuscript provides clarity, examines different endothelial types and different ways to get confluent cells and is therefore worthy.

Generally the results are displayed appropriately and the experiments appear sound. Summary data is shown throughout, showing individual experimental points in addition would make the results easier to interpret. More detail in the legends would also help. N numbers are mentioned in the methods but they should also be included in the legends as well as a description of what the error bars represent (SD, SEM etc). Similarly when the WPB numbers are quoted in the text the SD or SEM should also be included to allow readers to assess the veracity of the data.

Fig.1

The legend on the scale bar is really small and should be easier to read. Error bars represent SEM/SD? It would be helpful to have an image at 0h to compare to 100h. It us unclear which timepoint has been shown.

Fig.2

Summary data alone is shown. Individual experimental points would help interpretation (or this data should be shown as supplementary) B and D need error bars ideally.

Fig.3 and 4

Summary data alone is shown, individual data points need to be shown also. The definition of confluency is arbitrary, the authors need to give some idea as to how they delineated confluent vs non confluent with representative images. Microscopy of proteins present at the junctions such as VE Cadherin would make this clearer and allow the readers to gauge the effects and compare the cell types (the authors mentioned differences in cell size etc).

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Reviewer #1: No

Reviewer #2: No

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Quantitative Analysis of Weibel-Palade Bodies

PONE-D-22-04392R1

Dear Dr. Liu,

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,

Mária A. Deli, M.D., 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: All comments have been addressed

Reviewer #2: All comments have been addressed

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

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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 have revised some of the text of this manuscript. I agree with their comment that the findings of this paper, which is that the route to confluency does not impact the eventual steady state WPB count, can be of interest to other investigators when planning their experiments.

Reviewer #2: The presentation of the figures are improved and although the authors have not been able to use markers of confluency they have added caveats as to how the data was analysed and the associated implications of this. The primary data is available but is not included as supplementary data and this might be something that will make the findings of the work more impactful.

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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

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