Axl contributes to efficient migration and invasion of melanoma cells

Hanshuang Shao; Diana Teramae; Alan Wells

doi:10.1371/journal.pone.0283749

Peer Review History

Original SubmissionNovember 4, 2022
1 Feb 2023 Decision Letter - Suzie Chen, Editor PONE-D-22-30461Axl contributes to efficient migration and invasion of melanoma cellsPLOS ONE Dear Dr. Wells, 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 review carefully the concerns raised by both reviewers and address each point accordingly in your revised mansucript. Please submit your revised manuscript by Mar 18 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, Suzie Chen Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by a Merit Award from the Veterans Administration" We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This work was supported by a Merit Award from the Veterans Administration (AW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "A preliminary version of this manuscript has been uploaded to bioRxiv at " ext-link-type="uri" xlink:type="simple">https://www.biorxiv.org/content/10.1101/2022.05.02.490307v1" Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. 5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 6. 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. 7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 8. Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure. 9. 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. [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: Partly ******** 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: No ****** 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: The manuscript by Shao and colleagues describes investigation into the effect of AXL stimulation and level on the migration and invasion of melanoma cells. The authors demonstrate clearly that stimulation of Axl signalling with Gas6 increases melanoma migration and invasion in lines with Axl expression. Further, knockdown of Axl expression either using transient or stable means reduces melanoma migration and invasion, as does treatment with a specific inhibitor. While the data arising from the stable knockdown is novel, much of the other data presented in the manuscript is confirmatory. The manuscript overall would benefit from careful revision and editing. The experiments appear to have been conducted carefully with sufficient replication evident from the description. However, the authors should acknowledge the previous work of others in the manuscript on the topic at hand: Sensi M, et al. (2011). "Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase." J Invest Dermatol 131:2448–57. Use of shAXL to reduce invasion in matrigel/boyden chamber Use of R428 to reduce migration and invasion Tworkoski, K., et al. (2011). "Phosphoproteomic screen identifies potential therapeutic targets in melanoma." Mol Cancer Res 9(6): 801-812. Use of shAXL to reduce migration Pietrobono S, Anichini G, Sala C, Manetti F, Almada LL, Pepe S, Carr RM, Paradise BD, Sarkaria JN, Davila JI, Tofani L, Battisti I, Arrigoni G, Ying L, Zhang C, Li H, Meves A, Fernandez-Zapico ME, Stecca B. ST3GAL1 is a target of the SOX2-GLI1 transcriptional complex and promotes melanoma metastasis through AXL. Nat Commun. 2020 Nov 17;11(1):5865. doi: 10.1038/s41467-020-19575-2. Use of shAXL to reduce invasion in matrigel/boyden chamber Zhang X, Yang L, Szeto P, Abali GK, Zhang Y, Kulkarni A, Amarasinghe K, Li J, Vergara IA, Molania R, Papenfuss AT, McLean C, Shackleton M, Harvey KF. The Hippo pathway oncoprotein YAP promotes melanoma cell invasion and spontaneous metastasis. Oncogene. 2020 Jul;39(30):5267-5281. doi: 10.1038/s41388-020-1362-9. Epub 2020 Jun 19. PMID: 32561850 Use of shAXL to reduce invasion in matrigel/boyden chamber In summary, the data presented in Figure 2A and B, Figure 3A-D and Figure 4A-D has been previously published by others. Figure 2A is currently very confusing. It would be best to group the western blot images by cell line rather than antibody. Further, the figure legend states 10 nM EGF was used whereas the results text states 2 nM. Which is correct? There is currently no text in the results section that addresses the data in Figure 5. The reference to figures throughout the manuscript needs revision. Further, the labelling of Supplementary Figure 2 A, B - A, B, C and D is confusing and should be revised. Reviewer #2: Eight melanoma lines were evaluated for expression of EGFR and AXL family members. IgR3 and WM852 were then stimulated with Gas6 and EGF and evaluated for pAKT, pAXL, p38, pEGFR, and pERK. Knockdown of AXL blocked Gas6 induced pAKT in both lines.as well as a line lacking AXL (WM983b) although TYRO3 was present, indicating that AXL is a mediating of Gas6 induced Akt phosphorylation. WM852 showed stronger relative migration speeds induced by Gas6 or EGF compared to IgR3. With respect to invasion, IgR3 showed higher basal migration than WM852 but there was a similar absolute increase induced by Gas6. Basal migration and invasion of the two lines was reduced either by an AXL inhibitor or siRNA. The main claims of the paper are that AXL can mediate motility and invasion of melanoma cell lines. This has not been demonstrated previously for melanoma and thus adds to the body of knowledge for this disease. The authors should discuss the current knowledge regarding AXL mediating Gas6 induced migration/invasion in other tumor types. There are some concerns regarding selected claims noted below. Detailed protocols are not required but more details regarding specific reagents and the initial width in the scratch assay should be indicated. The manuscript is written clearly and well organized with no dual use concerns. Other comments: On page 5, there is reference to “all 4 MGP” and “all 4 VGP”. Figure 1 however does not clearly indicate 4 VGP lines – please correct the inconsistency. Is WM983B a VGP, MGP or neither? In referring to Figure 2 on page 5, it is stated” Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852.” However, the blot seems to clearly show increased pERK for IgR3 in response to EGF and possibly also for GM852. Similarly, though weaker, there is EGFR phosphorylation in response to EGF although it is claimed that there is not. The statement on page 6 in the middle : “These findings indicate that Axl signaling is cell-line specific” is confusing – one possibly interpretation is there are lines that express axl but do not respond to Gas6. Please clarify or remove. Catalog numbers for siRNAs, antibodies, transwells, growth media, growth factors (Gas6, EGF) and all other biological reagents should be provided. For Figure 3, please indicate the absolute basal migration speeds for all the lines. Does the smaller relative increase for IgR3 simply reflect a higher basal migration speed? The statement at the top of page 7 “…suggest that pAkt plays important role in Gas6-mediated enhanced migration of IgR3 and WM852” does not seem justified since no inhibitor of Akt phosphorylation was tested. Figure S2 legend refers only to panels A and B while the figure is labeled A- D. In the discussion it is stated that ASL siRNA eliminated Gas6 induced p38 phosphorylation – that does not seem supported by Figure 2 since there is little gas6 induced p38 phosphorylation, especially in IgR3. Quantitative analysis and statistics should be used to show this point. ****** 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: 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. https://doi.org/10.1371/journal.pone.0283749.r001
Revision 1
2 Mar 2023 Author Response Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Response: Please see the response below. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by a Merit Award from the Veterans Administration" We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This work was supported by a Merit Award from the Veterans Administration (AW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: It is now removed from the manuscript. Please amend to: "This work was supported by a Merit Award from the Veterans Administration (BX003368 to AW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 4. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "A preliminary version of this manuscript has been uploaded to bioRxiv at https://www.biorxiv.org/content/10.1101/2022.05.02.490307v1" Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. Response: The posted manuscript was neither peer-reviewed nor formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. 5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Response: All underlying data are presented in the manuscript and the Supporting Information files. There are no further data to post. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Response: Please state that all data are presented in the manuscript and the Supporting Information files. 6. 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. Response: Both cropped and the cognate uncropped blots are now provided in the Supporting Information files. 7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Response: We now provide these data in the revised manuscript to show that the invasion of IgR3 in 1% FBS medium is significantly higher than that in 0.1% FBS medium. 8. Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure. Response: We now address the data in Figure 5 in the revised text. 9. 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. Response: We have updated the references as suggested by the reviewers. 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 by Shao and colleagues describes investigation into the effect of AXL stimulation and level on the migration and invasion of melanoma cells. The authors demonstrate clearly that stimulation of Axl signalling with Gas6 increases melanoma migration and invasion in lines with Axl expression. Further, knockdown of Axl expression either using transient or stable means reduces melanoma migration and invasion, as does treatment with a specific inhibitor. While the data arising from the stable knockdown is novel, much of the other data presented in the manuscript is confirmatory. Comment: The manuscript overall would benefit from careful revision and editing. The experiments appear to have been conducted carefully with sufficient replication evident from the description. However, the authors should acknowledge the previous work of others in the manuscript on the topic at hand: Sensi M, et al. (2011). "Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase." J Invest Dermatol 131:2448–57. Use of shAXL to reduce invasion in matrigel/boyden chamber Use of R428 to reduce migration and invasion Isolated melanoma cell lines Tworkoski, K., et al. (2011). "Phosphoproteomic screen identifies potential therapeutic targets in melanoma." Mol Cancer Res 9(6): 801-812. Use of shAXL to reduce migration Yale isolated melanoma cell lines Pietrobono S, Anichini G, Sala C, Manetti F, Almada LL, Pepe S, Carr RM, Paradise BD, Sarkaria JN, Davila JI, Tofani L, Battisti I, Arrigoni G, Ying L, Zhang C, Li H, Meves A, Fernandez-Zapico ME, Stecca B. ST3GAL1 is a target of the SOX2-GLI1 transcriptional complex and promotes melanoma metastasis through AXL. Nat Commun. 2020 Nov 17;11(1):5865. doi: 10.1038/s41467-020-19575-2. Use of shAXL to reduce invasion in matrigel/boyden chamber A375 cell line and patient-derived melanoma cells. We actually cited this paper in ref #4 Zhang X, Yang L, Szeto P, Abali GK, Zhang Y, Kulkarni A, Amarasinghe K, Li J, Vergara IA, Molania R, Papenfuss AT, McLean C, Shackleton M, Harvey KF. The Hippo pathway oncoprotein YAP promotes melanoma cell invasion and spontaneous metastasis. Oncogene. 2020 Jul;39(30):5267-5281. doi: 10.1038/s41388-020-1362-9. Epub 2020 Jun 19. PMID: 32561850 Use of shAXL to reduce invasion in matrigel/boyden chamber Response: We apologize for these oversights. We now added these data and references with statement in the discussion. Comment: In summary, the data presented in Figure 2A and B, Figure 3A-D and Figure 4A-D has been previously published by others. Response: We include these data as part of the complete story as these are new data sets but refer to earlier works in the text. We used Gas6 to check if the interaction between Axl and EGFR is involved in the activation of Gas6-depependent of Axl in available melanoma cell lines. These data also confirmed that Gas6 functions in Axl positive melanoma cells. Comment: Figure 2A is currently very confusing. It would be best to group the western blot images by cell line rather than antibody. Further, the figure legend states 10 nM EGF was used whereas the results text states 2 nM. Which is correct? Response: We now reorganized Fig. 2A by cell line according to reviewer’s suggestion. For short stimulation of cells with EGF, we usually use a concentration of 10 nM. We mistyped the concentration of EGF in text but that is now corrected. Comment: There is currently no text in the results section that addresses the data in Figure 5. Response: We apologize for this oversight. We now address the data in Figure 5. Comment: The reference to figures throughout the manuscript needs revision. Further, the labelling of Supplementary Figure 2 A, B - A, B, C and D is confusing and should be revised. Response: Figure S2 now is reorganized, relabeled and renumbered as Figure S3 in the revised manuscript. Reviewer #2: Eight melanoma lines were evaluated for expression of EGFR and AXL family members. IgR3 and WM852 were then stimulated with Gas6 and EGF and evaluated for pAKT, pAXL, p38, pEGFR, and pERK. Knockdown of AXL blocked Gas6 induced pAKT in both lines.as well as a line lacking AXL (WM983b) although TYRO3 was present, indicating that AXL is a mediating of Gas6 induced Akt phosphorylation. WM852 showed stronger relative migration speeds induced by Gas6 or EGF compared to IgR3. With respect to invasion, IgR3 showed higher basal migration than WM852 but there was a similar absolute increase induced by Gas6. Basal migration and invasion of the two lines was reduced either by an AXL inhibitor or siRNA. The main claims of the paper are that AXL can mediate motility and invasion of melanoma cell lines. This has not been demonstrated previously for melanoma and thus adds to the body of knowledge for this disease. Comment: The authors should discuss the current knowledge regarding AXL mediating Gas6 induced migration/invasion in other tumor types. There are some concerns regarding selected claims noted below. Detailed protocols are not required but more details regarding specific reagents and the initial width in the scratch assay should be indicated. The manuscript is written clearly and well organized with no dual use concerns. Response: We thank the reviewer for the suggestion. We now discussed this in the part of discussion of our revised manuscript. The width of the wound scratch area is about 3 mm. It is now stated in the revised manuscript. Other comments: Comment: On page 5, there is reference to “all 4 MGP” and “all 4 VGP”. Figure 1 however does not clearly indicate 4 VGP lines – please correct the inconsistency. Is WM983B a VGP, MGP or neither? In referring to Figure 2 on page 5, it is stated” Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852.” However, the blot seems to clearly show increased pERK for IgR3 in response to EGF and possibly also for GM852. Similarly, though weaker, there is EGFR phosphorylation in response to EGF although it is claimed that there is not. Response: We apologize for our imprecise statements. As the labeling in Figure 1, there are three VGP cell lines and five MGP cell lines; we now corrected this in the revised manuscript. For the reference to Figure 2 on page 5, we stated that “Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852” referred to no significant pERK increase when cells are treated only by Gas6. The reviewer is correct in that EGF treatment significantly increased the pERK which has been mentioned on page 6. We also stated that “the EGFR was functioning as shown by phosphorylation when exposed to 10 nM EGF for 10 min” on page 6. This meant that the EGF treatment triggers the phosphorylation of EGFR in both IgR3 and WM852 cells. It is stronger in WM852 and weaker in IgR3. These statements are now clarified. Comment: The statement on page 6 in the middle: “These findings indicate that Axl signaling is cell-line specific” is confusing – one possibly interpretation is there are lines that express axl but do not respond to Gas6. Please clarify or remove. Response: We thank the review for pointing out this issue. We now deleted the statement in the revised manuscript. Comment: Catalog numbers for siRNAs, antibodies, transwells, growth media, growth factors (Gas6, EGF) and all other biological reagents should be provided. Response: We now provide catalog numbers for all reagents. Comment: For Figure 3, please indicate the absolute basal migration speeds for all the lines. Does the smaller relative increase for IgR3 simply reflect a higher basal migration speed? Response: The reviewer is correct in that IgR3 presents much high basal migration. We now updated Figure 3C with relative migratory units instead of normalized data to more clearly show this. Comment: The statement at the top of page 7 “…suggest that pAkt plays important role in Gas6-mediated enhanced migration of IgR3 and WM852” does not seem justified since no inhibitor of Akt phosphorylation was tested. Response: We thank the reviewer for pointing out this misstatement. We now corrected it to “suggest that Axl plays an important role in Gas6-mediated enhanced migration of IgR3 and WM852. Comment: Figure S2 legend refers only to panels A and B while the figure is labeled A- D. Response: We thank the reviewer for noting this omission. We now reorganized this figure and corrected the figure legend as shown in figure S3 in the revised manuscript. Comment: In the discussion it is stated that ASL siRNA eliminated Gas6 induced p38 phosphorylation – that does not seem supported by Figure 2 since there is little gas6 induced p38 phosphorylation, especially in IgR3. Quantitative analysis and statistics should be used to show this point. Response: We thank the reviewer for noting the lack of significance of this and have corrected this in the revised manuscript. Attachments Attachment Submitted filename: Response Letter R1.pdf https://doi.org/10.1371/journal.pone.0283749.r002
15 Mar 2023 Decision Letter - Suzie Chen, Editor Axl contributes to efficient migration and invasion of melanoma cells PONE-D-22-30461R1 Dear Dr. Wells, 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, Suzie Chen 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 ******** 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: (No Response) Reviewer #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: (No Response) ****** 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: (No Response) Reviewer #2: (No Response) ****** 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: (No Response) Reviewer #2: (No Response) ****** 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: (No Response) Reviewer #2: (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 #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0283749.r003
Formally Accepted
20 Mar 2023 Acceptance Letter - Suzie Chen, Editor PONE-D-22-30461R1 Axl contributes to efficient migration and invasion of melanoma cells Dear Dr. Wells: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Suzie Chen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0283749.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .