PONE-D-20-12629

Adenomatous polyposis coli-binding protein end-binding 1 promotes hepatocellular carcinoma growth and metastasis

PLOS ONE

Dear Dr. Yokoo,

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.

Two experts have reviewed the manuscript and foudn that the study needs to be strengthened with more clear presentatinos via more critical statistic analysis, controls, and WB or images in higher qualities.

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Jung Weon Lee, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

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: In the manuscript, the authors described a study to determine the clinical significance of adenomatous polyposis coli-binding protein end-binding 1 (EB1) in hepatocellular carcinoma (HCC). The authors performed various experiments and concluded that elevated expression of EB1 promotes tumor growth and metastasis of HCC, suggesting using EB1 as a HCC biomarker. While evidence from multiple experiments seems supportive of the conclusion, the sample sizes are small in most of the experiments, and the statistical significance of many tests seems marginal.  A few comments:

[Page 9, 10; Table 1] For numerical variables like age and tumor size, it would be good to provide standard deviation in addition to the mean values.  For categorical variables, the p-values calculated seem different from those by Fisher's exact tests.

[Page 12; Fig 2c, d, and e]

The sample size may be too small to draw meaningful conclusion, and it would be good to state the mean and standard deviation values for proliferation, migration, and invasion in the manuscript.

[Page 12; Fig 3b, c, and d]

The sample size is too small, and the p-values may not be valid.  It might be better to include more biological replicates.

[Page 13; Fig 4]

Again the sample size is too small to draw any conclusion with statistical significance.  More samples would be recommended.

[Page 14; Fig 5b]

While the list of up-regulated genes is interesting, it would be of interest to analyze any potential functional or pathway enrichment.

Reviewer #2: The manuscript by Aiyama and coworkers confirms the involvement of EB1 in HCC by showing that its expression is associated with poor survival and high recurrence rate. Additionally, in vitro and in vivo experiments show involvement of EB1 in proliferation, migration, invasion and tumor growth. The study appears solid, but there are a few issues, mainly regarding lack of information in the methods, that should be addressed.

1) Was the assesment of staining done just by eye or was imageJ or equivalent used ? Where is the 30% staining cutoff based on (Orimo 2008 study used 50%?!), and how was this exactly determined?

2) For all-in vitro experiments information is lacking regarding technical replicates and the number of independent experiments performed.

3) Is it certain the b-actin mRNA remains stable upon EB1 knockdown or overexpression ? It is standard to use 2-3 genes for qPCR normalization, not just 1.

4) Same for WB normalization, is b-actin proven to remain stable? Has normalization also been done using a whole protein stain?

5) In the migration assays, was the membrane of the transwell plate completely uncoated? And for invasion, what was the volume of matrigel used to coat the transwells?

6) Fiugre 2a: to choose HLF, HLE and HuH7 appears a bit random as all but JHH4 and HepG2 appear suitable. Please rephrase line 220.

7) Why was specifically HuH7 chosen fort he knockout experiments? Please clarify.

8) All data in figure 3 should be shown in comparison to the original HuH7 cells, especially to confirm efficient knockout.

9) Line 300 : Unclear why SLCO1B3 is discussed and for instance NPTX2 and HMSD not. Additionally, the data from the microarray should be validated by at least qPCR before these can be considered reliable enough to thoroughly discuss in line 341 to 359.

10) Line 363-364 is unclear, this statement requires further explanation.

11) Overall, the Western blot images quality is quite poor and higher resolution images are needed.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-12629R1

Adenomatous polyposis coli-binding protein end-binding 1 promotes hepatocellular carcinoma growth and metastasis

PLOS ONE

Dear Dr. Yokoo,

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.

==============================

The rpevious reviewers found that the revised manuscript wa simproved but certain points should be explained further for the publucation.

==============================

Please submit your revised manuscript by Oct 10 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Jung Weon Lee, Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

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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: Partly

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 have addressed most of my comments. One key remaining concern is that for most of the statistical analyses, the sample sizes are small due to limited number of replicates (n=3). Although the p-values are often significant, the power of the tests could be small. This may be addressed by stating the limitations of the small sample sizes used in statistical tests, as part of the discussion section.

Reviewer #2: All of my comments have been adequately addressed. However, it would be appreciated if the responses to my comments #1 (explanation of how immunostainings were scored and the cut-off value obtained) and #5 (the concentration of Matrigel used) could also be added to the method section of the manuscript.

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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

[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.

Adenomatous polyposis coli-binding protein end-binding 1 promotes hepatocellular carcinoma growth and metastasis

PONE-D-20-12629R2

Dear Dr. Yokoo,

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.

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Kind regards,

Jung Weon Lee, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: