PONE-D-20-06136

Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers

PLOS ONE

Dear %Dr. Haglund%,

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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: 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: Yes

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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: No

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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: The manuscript focuses on the interaction between IGF1R and PCNA in various tumors and cancer cells lines, and its association with overall survival. Most of the tumors exhibited IGF1R/PCNA colocalizations. Furthermore, this type of colocalization is minimized in tumors with poor treatment response, as well as in metastatic lesions. The manuscript is written well, however, few revisions should be made to improve the overall quality.

Minor revisions:

1. In DNA Fiber assay, CidU should be changed to CldU throughout the manuscript, and in figures.

2. Scale in µm should be included in all figures with microscopy images.

3. Figures 4 and 5: fgi should be changed to fig

4. The quality of all figures appear to be low and the text within the figures is not legible. The figures should be at least 300dpi. Especially in figure 6, the images and graphs are very hard to see.

5. In figure 5, the signals for IGF1R are too saturated. For the text “Though no change in co-IP protein levels was detect in IGF1R after HU treatment, PCNA was both mono-ubiquitinated and co-immunoprecipitated with the E3 ubiquitin ligase Rad18 and HLTF (Figure 5C),” the corresponding figure should be changed to “Figure 5D”.

6. “Subsequent fluorescent PLA showed that the colocalization of IGF1R and PCNA was predominantly nuclear in HeLa cells (Figure 5D).” The figure should be “Figure 5C” here, which corresponds to immunofluorescence. In this figure, color for IGF1R and PCNA should be different to clearly visualized for colocalization.

Reviewer #2: In this manuscript, authors evaluated the role of nuclear IGF1R and its correlation with poor outcomes in cancer. Mechanistically, in situ proximity ligation assay identified frequent IGF1R and PCNA co-localization across many cancer types. While IGF1R/PCNA co-localization was found to be increased frequently in tumor cells versus adjacent normal tissue as well as in areas with dysplasia and invasion, the interaction was frequently lost in tumors with poor response to neo-adjuvant treatment and most metastatic lesions. Additionally, in clinical samples of serous ovarian carcinomas and oropharyngeal squamous cell carcinomas, stronger IGF1R/PCNA co-localization was significantly associated with better overall survival. IGF1R activation also rescues stalled DNA replication forks, but only in cancer cells with baseline IGF1R/PCNA interaction. Overall, authors summarize that cancer cells may utilize IGF1R phosphorylation of PCNA to increase DNA damage tolerance, but this mechanism is frequently lost with tumor progression.

• The manuscript clearly lacks clarity and mostly confusing. A clear explanation of the results and its significance in discussion is needed in the revised manuscript.

• There are numerous spelling mistakes in the manuscript. Authors have to completely re-check for these small error. Few are highlighted below.

• Line 69: PCNA

• Line119: for in

• Line 147: IGF1R

• Include scale bars in figure 1 and 2.

• Conclusion for this manuscript is achieved mostly by PLA assay. Authors should use additional methods or ways to confirm their result. More validation of TLS pathways is needed to confirm the conclusion.

• Apart from the nuclear PLA signal between IGF1R and PCNA, there are also some signals in cytoplasm. It will be helpful for the readers if authors can discuss about the cytoplasmic PLA signal’s significance.

• Line 153 to 163 corresponds to which figure? Supplementary figure 1 to 5 is not cited in the results section.

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Reviewer #1: No

Reviewer #2: No

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Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers

PONE-D-20-06136R1

Dear Dr. %Haglund%,

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. However, there are some typographical errors that needs to be addressed before publication. Please see below for the reviewer's comments.

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.

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Kind regards,

Komaraiah Palle, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Although minor revisions pertaining to spellings and grammar were edited well throughout the text, following typographical errors were observed and need to be edited.

1. In the Supplementary Table 1, Spelling of ‘skeletal’ in 18th row to be corrected.

2. In the Supplementary Information on Materials and Methods, 17th line, ‘the slides was…’ to be corrected to ‘the slides were…’

3. In the Supplementary Figure 10, ‘CidU’ to be corrected to ‘CIdU’

4. In the Supplementary Figure 12, the words, ‘significant and treatments’ are wrongly spelled.

5. The word DNA polymerase eta is mentioned differently (ETA, Eta and eta in text and figures). The notation should be uniform throughout the manuscript.

Overall, the clarity and flow of content is better in the revised draft. The manuscript is as per the standards of the Journal and hence can be accepted after verifying and editing the minor errors.

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

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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 #3: Yes

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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

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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 #3: Yes

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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: Review comments on the revised manuscript (PONE-D-20-06136R1)

submitted to PLOS ONE Journal

In the revised manuscript entitled, “Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers” the author(s) have incorporated the changes appropriately as per the suggestions of the reviewers. The text is thoroughly revised to improvise results and discussion sections while citing or editing the figure and table numbers at the relevant places. The authors have attempted to incorporate additional experimental results and emphasized on elaborating the significance of the results obtained in the discussion as suggested by one of the reviewers. The resolution and format of the figures has also been enhanced as commented.

Although minor revisions pertaining to spellings and grammar were edited well throughout the text, following typographical errors were observed and need to be edited.

1. In the Supplementary Table 1, Spelling of ‘skeletal’ in 18th row to be corrected.

2. In the Supplementary Information on Materials and Methods, 17th line, ‘the slides was…’ to be corrected to ‘the slides were…’

3. In the Supplementary Figure 10, ‘CidU’ to be corrected to ‘CIdU’

4. In the Supplementary Figure 12, the words, ‘significant and treatments’ are wrongly spelled.

5. The word DNA polymerase eta is mentioned differently (ETA, Eta and eta in text and figures). The notation should be uniform throughout the manuscript.

Overall, the clarity and flow of content is better in the revised draft. The manuscript is as per the standards of the Journal and hence can be accepted after verifying and editing the minor errors.

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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 #3: No