PONE-D-23-05403Ligand-Specific Regulation of Transforming Growth Factor Beta Superfamily Factors by Leucine-rich Repeats and Immunoglobulin-like Domains ProteinsPLOS ONE

Dear Dr. Hedman,

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. 

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ACADEMIC EDITOR: Both reviewers raised concerns about: 1) the lack of data on expression of the co-receptors following Dox treatment and how this relates to physiological levels, and 2) inappropriate statistical analyses. These two points muse be addressed with additional data and statistical analyses. Changes to the interpretation of results may be required as a result. Please address the more minor concerns as well.

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PLOS ONE

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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: No

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: No

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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: No

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: 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 their manuscript, Abdullah et al. the regulation of TGFβ signaling by leucine-rich repeats and immunoglobulin-like domains (LRIG) using Lrig-null mouse embryonic fibroblasts (MEFs) with a doxycycline-inducible LRIG1, LRIG2, or LRIG3 overexpression system. Cells were treated with different TGFβ ligands, after treatment, SMAD1/5/8 and SMAD3 phosphorylation, as well as Id1 expression was assessed.

Major points:

1) The authors do not show LRIG1, 2, and 3 protein expression levels before and after doxycycline-induction in their cell system and its correlation with normal/basal levels of LRIG proteins in wild-type MEFs cells. The data are not robust. SMAD-3 phosphorylation is weak in most of the treatments in wild-type cells, however, there is stimulation in the mutant cells treated with or without doxycycline. The authors should explain this discrepancy to validate the use of these cells to study SMAD phosphorylation dependant on LRIGs proteins.

2) The statistical analysis are not correct in any of the figures. The authors performed t-tests in an experimental design with 2 variables. Two-way ANOVA should be performed.

3) The authors used different concentrations of doxycycline to analyze LRIG1 and LRIG3 roles after GDF7 stimulation, however, the authors do not show these raw data neither how the different doses of doxycycline affect protein levels of LRIG1 or LRIG3 (Fig. 3).

4) It is not clear how robust and reproducible these data are. Experiments should be graphed using individual points, where each point correspond to one independent experiment in all figures.

5)It is not a clear if Id1 stimulation depends on LRIG proteins, there is dose-response effects in absence of doxycycline (Fig. 5).

Minor comments:

1) Colors and font sizes should be consistent across the figures.

2) Protein and gene nomenclature must be modified: proteins should be written in capital letters and genes in lower-case letters and italicized.

3) Graphs and statistical analysis were done with two different version of GraphPad. Please, clarify.

4) Immunofluorescence images are not available, only the quantification is plotted. A supplementary figure needs be added.

Reviewer #2: Abdullah et al. examined signaling of various TGFbeta ligands through the LRIG1-3 co-receptors in murine MEFs. This represents an extension of earlier work by this group. Overall, the manuscript is straightforward and easy to read. Most of my comments are minor in nature. The only ‘major’ concern (#1 below) is the level of expression of the co-receptors when over-expressed, which was not reported (see below). It is possible that additional experiments will be required to address this concern.

Specific comments:

1. The authors do not show expression of the different receptors following Dox treatment. It is important to show the extent to which each receptor was expressed relative to what is seen in wild-type MEFs. The authors imply that the expression level was or might have been supraphysiological (line 238), but they do not show data that speak to this issue. They should. The data in Fig. 3 show (at least for GDF7) that the effect of LRIG1 and LRIG3 on Smad1/5 activation is dependent on the extent of co-receptor expression. We need to see if any of the potentiation (by LRIG3) or attenuation (by LRIG1) occurs when the proteins are expressed at or near physiological levels. The authors claim to address concerns about supraphysiological expression starting on line 238, but this reviewer was not persuaded by their argument. We need to see protein expression and how it relates to what is seen in wild-type MEFs.

2. Line 19: ‘dependent’ here and elsewhere (‘dependency’, line 184, 233) is not the right word. If something is dependent on something else, it requires it. That is not the case here. LRIG impacts, regulates, influences, etc. signaling by some TGFbeta ligands, but in none of the cases reported here did their signaling depend on any of the LRIG proteins. Please replace the word ‘dependent’ or ‘dependency’ with more accurate terminology.

3. Line 38: While there may be 33 genes that encode TGFbeta ligand subunits, there are many heterodimers. Therefore, the number of ligands is likely to be far greater than 33.

4. Line 46: Based on work from the Hill lab, it is more accurate to say that Smad complexes accumulate rather than translocate into the nucleus.

5. Line 119: Here and in the associated figures, why normalize with actin rather than with total (unphosphorylated) Smads?

6. Line 155: Provide references for the reported EC50s. Are these EC50s specific for these ligands in MEFs? It is surprising that 100 ng/ml was used as the EC50 for GDF11, for example. This seems very high. Also, given the receptor expression in these cells, it is surprising that activin A did not stimulate pSmad3. Perhaps 2 ng/ml is too low in these cells. Ideally, the authors should have done dose-responses for all the ligands used here in these cells. In lieu of asking for these data, I think the authors need to do a better job of justifying the concentrations they used (with references). They might also add some text (to the Discussion) acknowledging that they might have been able to assess LRIG regulation of the other ligands had the latter been tested at different (higher?) concentrations.

7. Line 156: There is no description of western blotting for pSmad1/5 in the Methods. At present, western blotting is limited to pSmad3. Please update the Methods.

8. Line 159: The GDF11 data are not convincing here (they are more convincing is subsequent experiments). In Suppl. Fig. 1, only TGFbeta1 convincingly stimulates pSmad3. I don’t see a difference between GDF11 and GDF15 on any of the blots. Also, in the figure, activin should be labeled activin A.

9. Line 170: For the residual Lrig expression, the authors can (and should) look for sequence reads in the floxed exons. If the recombination was complete, there should be no reads for these exons. This information can and should be reported. This seems particularly important for Lrig1.

10. Line 179: What was the rationale for using IF for pSmad1/5 vs. IB for pSmad3? Why different methods for the two?

11. Lines 201-217 (and Figure 5): There is no discussion/mention of the experiment with LRIG2 for BMP2. Also, why wasn’t LRIG2 tested with the other ligands in this experiment?

12. Line 271: I am not convinced that there is a qualitative difference between LRIG2’s effects on BMP2 vs. BMP4. In figure 1, the difference really looks quantitative. I am not certain the correct statistics were used in these experiments. The Methods (line 142) reference t-tests. Given the nature of these experiments (and in Figs. 2, 4, and 5), the authors should be using two-way ANOVAs (or a non-parametric test if the variances are not equal or if they do not log transform their data). In Fig. 1, there is a clear main effect of Dox for BMP4 and BMP6 (maybe even for TGFbeta1). That is, it is clear that LRIG2 is also potentiating their responses as it does for BMP2. The authors should employ the correct statistics and adjust their discussion, as necessary. The correct stats may impact the interpretation of the data in Fig. 4 (BMP9) as well.

13. Line 272-273: The differential effect of LRIG1 and LRIG3 on GDF7 signaling is arguably the most novel and interesting of the study. It is disappointing that no mechanistic insight is provided. According to Fig. 6, GDF7 signals preferentially via ALK6. But ALK6 is expressed at low levels in these cells (indeed, as in most cells). Might LRIG1 and LRIG3 differentially affect GDF7 signaling via ALK3 or even ALK2?

14. Lines 285-286: Is the discussion here based on the relative abundance of these receptors (at least at the mRNA level) in these cells? If so, that should be stated explicitly, as there are no functional data here on the roles of these different type I receptors.

15. Figure 3: Y axis. SMAD is all upper case here but is written as Smad elsewhere. Be consistent.

16. Figure 6: Activin A should be repositioned. It only used ALK4. It does not signal via ALK7.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-23-05403R1Ligand-Specific Regulation of Transforming Growth Factor Beta Superfamily Factors by Leucine-rich Repeats and Immunoglobulin-like Domains ProteinsPLOS ONE

Dear Dr. Hedman,

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.

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

ACADEMIC EDITOR:

The authors were responsive to previous feedback. There are just a few minor issues remaining to be addressed. Please see the reviewer comments. 

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

Please submit your revised manuscript by Aug 19 2023 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,

Daniel J. Bernard

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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

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

Reviewer #1: No

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: 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 #1: Yes

Reviewer #2: 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 #1: In their revised manuscript, Abdullah et al. addressed most of the previous comments from this reviewer. However, the authors are missing some relevant information in the ms.

1) Post-hoc comparisons used in their statistical analysis (two-way ANOVA) needs to be added.

2) Not all the supplementary figures (S1-S9) are discussed and referred in the text. For example, references to each supplementary figure must be added in lines 211, 218, and 286.

Reviewer #2: The authors were highly responsive to my prior feedback. It is unfortunate that the results still lack mechanistic insight, but the basic phenomena are interesting and worth reporting.

I have a few remaining minor comments:

1. There are 9 supplementary figures. These are not presented in order (i.e., reference is made to supplementary figure 9 before 1; 3 is referenced before 2) or are not referenced at all (supplementary figures 4, 5, 6, 7, and 8). Ideally, all supplementary figures should be referenced in the text and/or in the legends, and should be referenced in order of presentation.

2. Line 82: ‘college’ should be ‘colleague’

3. Lines 214-218: no figure is referenced in association with the described data. I think this may relate to supplementary figure 7 (though see my first comment).

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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.

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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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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.

Ligand-Specific Regulation of Transforming Growth Factor Beta Superfamily Factors by Leucine-rich Repeats and Immunoglobulin-like Domains Proteins

PONE-D-23-05403R2

Dear Dr. Hedman,

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.

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Daniel J. Bernard

Academic Editor

PLOS ONE