Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway

Xianxian Wu; Boram Lee; Lingxiang Zhu; Zhi Ding; Yin Chen

doi:10.1371/journal.pone.0231990

Peer Review History

Original SubmissionJanuary 11, 2020
6 Feb 2020 Decision Letter - Hong Wei Chu, Editor PONE-D-20-00929 Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway PLOS ONE Dear Dr. Chen,, 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 by the reviewer. We would appreciate receiving your revised manuscript by Mar 22 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Hong Wei Chu 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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. At this time, we ask that you please provide the product numbers and lot numbers of the A. fumigatus extracts purchased from GREER (Lenoir, NC), the inhibitors/neutralizzing antibodies and primary antibodies used in this study. 3. Thank you for stating in your Funding Statement: "The study was supported partly by NIH grant AI39439. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. 4. 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. 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 6. 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. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 cells response to AFE in MUC5AC transcripts are more than 10 times higher in the 24 hours time-point. Please clarify why 6 hours time-point was chosen. Is the concentration of AFE physiologically relevant during fungal infections? Please clarify why these doses were chosen. It was mentioned earlier that the 6 hrs time-point was selected. Please clarify why showing the 24 hrs time-point in figure 1? Please quantify MUC5AC and MUC5B protein levels for figure 1. What are the diluents of EGFR inhibitors? Do cells survive for 24 hrs after the EGFR inhibitor treatment? An LDH cytotoxicity assay will determine how toxic these inhibitors are to the cells. At which time-point cells were stained in figure 5 E? A Dot Blot can be performed for figure 2 to determine the MUC5AC and MUC5B protein levels reduction in the presence of EGFR inhibitors. LDH cytotoxicity assay should be performed for figure 3 to determine the toxicity of incubating cells with trypsin for 6 hrs. Graph labels are not clear figure 3 B-C. What percentage of DMSO was used in figure 4? Is this amount of DMSO toxic to cells? LDH cytotoxicity assay will answer this question. Please clarify why MUC5B data is not shown in figure 5? Please highlight the significance of this work and how it contributes to the asthma research field in the discussion section. ****** 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: Yes: Nastaran Mues [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0231990.r001
Revision 1
20 Mar 2020 Author Response Dear reviewer: Thank you for your advice, it was very helpful for me. Here are my answers. Reply=R Response for first review: 1. Please ensure that your manuscript meets PLOS ONE's style requirements. R: Yes, I have checked. My manuscript meets PLOS ONE's style requirements. 2. At this time, we ask that you please provide the product numbers and lot numbers of the A. fumigatus extracts purchased from GREER (Lenoir, NC), the inhibitors/neutralizzing antibodies and primary antibodies used in this study. R: The product sheet is below. 3. Thank you for stating in your Funding Statement: "The study was supported partly by NIH grant AI39439. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. R: Yes, I have corrected it in my cover letter and I have changed my Funding sourse. 4.About the WB original data. In your cover letter, please note whether your blot/gel image data are in Supporting. Information or posted at a public data repository. R: I have provided my original blot data via Submission System and corrected it in my cover. letter. 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. R: Here is my ORCID iD: https://orcid.org/0000-0002-2191-6873. 6. 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. R: Yes, I have already corrected it. 7、Reviewer #1: The cells response to AFE in MUC5AC transcripts are more than 10 times higher in the 24 hours time-point. Please clarify why 6 hours time-point was chosen. R: For the mechanistic study, the early event is usually driven by a few immediate signaling pathways that are easy to dissect. When the exposure time goes on, secondary or tertiary events triggered by initial signaling may complicate the overall data interpretation. Therefore, we tend to choose the time points as early as the responses are demonstrated. 8、Is the concentration of AFE physiologically relevant during fungal infections? Please clarify why these doses were chosen. R: We tested the dose response and found that this dose can induce high levels of mucin expression without outright cell death. In reality, the level of AFE exposure has been difficult to establish as its direct measurement is lacking. Nonetheless, AFE exposure model will lead to the discovery of mechanistic basis of Aspergillus induced illnesses. 9、It was mentioned earlier that the 6 hrs time-point was selected. Please clarify why showing the 24 hrs time-point in figure 1? Please quantify MUC5AC and MUC5B protein levels for. figure 1. R: The 24hr time point is shown to demonstrate that this effect is time-dependent. The quantified protein levels are shown in Fig 1F. 10、What are the diluents of EGFR inhibitors? R: DMSO. 11、Do cells survive for 24 hrs after the EGFR inhibitor treatment? An LDH cytotoxicity assay will determine how toxic these inhibitors are to the cells. R: Yes, cells still survive. Related data is in S1_File. 12、At which time-point cells were stained in figure 5 E? R: I think you mean Fig 5A, cells were pre-treated with 50 uM BAPTA-AM for 30 min and. then used 5ul Fluo-4/AM in 1ml OPTI-MEM for another 30 min. After, wash 2x with OPTI-MEM (DMEM/HEPES/no phenol) and let it sit in fresh DMEM for another 30 minutes to allow the ester to cleave. At last, they were treated AFE for 3, 6,9, 10 min. 14、LDH cytotoxicity assay should be performed for figure 3 to determine the toxicity of incubating cells with trypsin for 6 hrs. R: Related data is in S1_File. 15、Graph labels are not clear figure 3 B-C. R: Thank you, I have corrected this. 16、What percentage of DMSO was used in figure 4? Is this amount of DMSO toxic to cells? LDH cytotoxicity assay will answer this question. R: The maximum percentage is 0.3% for U0126 inhibitor in this experiment. Related data is in S1_File. 17、Please clarify why MUC5B data is not shown in figure 5? R: Because 5B induction by AFE treatment is a little bit low, we didn’t show it at first, but I. have added this data in Fig 5C. 18、Please highlight the significance of this work and how it contributes to the asthma research field in the discussion section. R: Please see the highlight part in revised manusription. Response to second review: 1) Please provide additional information about each of the cell lines used in this work, including any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. R: I have add relevant information in Cell line section. 2) To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional. information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. R: I have corrected my statistical analyses in your Methods section. 3) At this time, we ask that you please add scale bars to the microscopy images presented in Figures 1. and 5 and refer to the scale bar in the corresponding Figure legend. R: Yes, I have add the scale bar analyses in Figures 1. and 5. Product name Product number Lot number Aspergillus fumigatus XPM3C3A25 231795 AG1478 658548-1MG D00108234 U0126 662005-1MG D00122919 BIBX 1382 324832-5MG B77319 neutralizing anti-EGFR Ab 05-101 0120201A1 Raf-1 inhibitor 553003 Sorafenib S-8502 BSF-106 PMSF P7626-5G BCCB9214 Glutathione reduced ethyl ester G1404-25MG BCCB5246 PORCINE Trypsin 85450C-25MG 12N438AR1C EGF 01-407 DAM1687496 BAPTA/AM 196419-25MG 3278031 pERK1/2 (T202/Y204) Ab 9101S 15 pRaf-1 (Ser289/296/301) Ab 9431S 2 anti-PAR2 neutralizing (SAM11) Ab SC-13504 C0713 anti-MUC5B (H-300) Ab SC -20119 F0611 anti-pEGFR (Y1173) Ab SC -12351 E2813 anti-EGFR (1005) Ab SC -03 H1007 anti-pTyr (py99) Ab SC -7020 L1106 β- ACTIN (C4) Ab SC -47778 H0515 anti-MUC5AC Ab MS-145-PI 145P1405I EnzChek® Protease Assay Kit E6638 1445257 RAS GTPASE PULLDOWN Kit PI16117 PA199259 Promega* Dual-Glo* Luciferase Assay PR-E2940 0000303692 Cell Proliferation Assay Kit G3580 0000328878 Fluo-4/AM F14201 2165131 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0231990.r002
6 Apr 2020 Decision Letter - Hong Wei Chu, Editor Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway PONE-D-20-00929R1 Dear Dr. Chen, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Hong Wei Chu Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0231990.r003
Formally Accepted
10 Apr 2020 Acceptance Letter - Hong Wei Chu, Editor PONE-D-20-00929R1 Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway Dear Dr. Chen: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Hong Wei Chu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231990.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 .