Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesis

Xiao-Hong Li; Jie-Jun Fu; Xiao-Juan Shi; Yun-Na Zhang; Min Shao; Shao-Jie Yue; Chen Li; Zi-Qiang Luo

doi:10.1371/journal.pone.0289530

Peer Review History

Original SubmissionDecember 19, 2022
21 Feb 2023 Decision Letter - Yi Cao, Editor PONE-D-22-33593Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesisPLOS ONE Dear Dr. Luo, 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 submit your revised manuscript by Apr 07 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. 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[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: 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 ****** 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 this manuscript, Li and colleagues have tried to elucidate the mechanism of phosphatidylcholine biosynthesis under over-activated glutamate/NMDA signaling conditions. Most of what is described in the paper has been previously described in the literature, with the exception possibly being of cytoplasm translocation of Sp1 after NMDA treatment. However, the data from immunofluorescence staining cannot support their wording. Other more readily addressable issues are listed below. 1. (Line 194: Reporter plasmid of CCTα promoter PGL3-CCTα (-1893/+71) …) There is no data presented using this plasmid. Which species (human or rat) of the CCTα promoter sequence is used in reporter assay? Since the DNA sequence between humans and rats is different, the details of Sp1 binding sites within the CCTα promoter should be provided. 2. The data confirming Sp1 overexpression is lacking. 3. (Figure 5-7) The glutamate effects on Sp1 should be examined. Also, rescue experiments should be performed using the NMDAR blocker (MK-801). 4. (Line 285: These results indicated that NMDAR activation inhibited the nuclear translocation of Sp1 …) No data supported this wording unless the protein level of Sp1 is increased or maintained in the cytoplasm fractionation. 5. (Figure 7) The Sp1 immunofluorescence data of NMDA treatment is out of focus. The lens focus is on the mitotic cells. Sp1 is disassociated with mitosis DNA, as described in the previous literature. Also, the quality of image data should be improved and present higher magnification images. 6. Typo: Figure 2B, Glutamae -> Glutamate ****** 6. 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Revision 1
8 May 2023 Author Response Responses: Thank you for your reviews. 1.Reporter plasmid of CCTα core promoter PGL3-CCTα (-169/+71) and pRL-SV40 empty vector with luciferase labeled promoter were from Mallampalli R.K. professor of the university of Iowa. The murine CCTα core promoter fragment (-169/+71) was generated by PCR using the following primers: 5’-TTGTGTGTTTTCACCCCTTATG-3’ (left) and 5’-TCAACTCCTCCAGGCTCCGGT-3’ (right) as described in literature. The core promoter of CCTα gene was localized to a region between -169 and +71 bp, which exhibited strong basal activity comparable with a positive control that contains the simian virus 40 promoter. (Ryan AJ, Fisher K, Thomas CP, Mallampalli RK. Transcriptional repression of the CTP:phosphocholine cytidylyltransferase gene by sphingosine. Biochem J. 2004 Sep 1;382(Pt 2):741-50. doi: 10.1042/BJ20040105. PMID: 15139854; PMCID: PMC1133833.); (Mallampalli RK, Ryan AJ, Carroll JL, Osborne TF, Thomas CP. Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter. Biochem J. 2002 Feb 15;362(Pt 1):81-8. doi: 10.1042/0264-6021:3620081. PMID: 11829742; PMCID: PMC1222362.) We conducted cell experiments using these plasmids in A549 cells and did obtain such result. We did not consider the issue of species at that time, but it was indeed a very thought-provoking issue. Therefore, we have decided to remove this result from the article to avoid unnecessary misunderstandings, but this does not affect our conclusion. As the reviewer pointed out, the impact of NMDAR activation on Sp1 nuclear translocation is the novelty of our study. Sp1 is part of a family of approximately 20 proteins that is characterized by having a DNA-binding domain that has 3 Krüppel-like zinc fingers (KLF). Studies have reinforced the central role for Sp1 in activating and promoting the transcription of CCTα gene. (Sugimoto H, Banchio C, Vance DE. Transcriptional regulation of phosphatidylcholine biosynthesis. Prog Lipid Res. 2008 May;47(3):204-20. doi: 10.1016/j.plipres.2008.01.002. Epub 2008 Feb 6. PMID: 18295604.) Banchio et al. demonstrated that increased binding of the transcription factor Sp1 to the proximal promoter of CCTα is responsible for increased transcription during the S phase. The Sp1 binding element present in position -67/-62 is essential for activation, and the Sp1 site in position -31/-9 is required to enhance transcription. (Banchio C, Schang LM, Vance DE. Activation of CTP:phosphocholine cytidylyltransferase alpha expression during the S phase of the cell cycle is mediated by the transcription factor Sp1. J Biol Chem. 2003 Aug 22;278(34):32457-64. doi: 10.1074/jbc.M304810200. Epub 2003 Jun 6. PMID: 12794070.) Bakovic et al. also demonstrated that Sp1 binding site 3 (-148/-128) and 1 (-31/-9) might activate or repress transcription of CCTα depends upon the cellular background and Sp1 binding site 2 (-88/-50) would confer a positive regulation independent of the cell context. (Bakovic M, Waite KA, Vance DE. Functional significance of Sp1, Sp2, and Sp3 transcription factors in regulation of the murine CTP:phosphocholine cytidylyltransferase alpha promoter. J Lipid Res. 2000 Apr;41(4):583-94. PMID: 10744779.) 2.We have supplemented the data confirming Sp1 overexpression, as shown in figure 4 (D, E). 3.In the study, we have demonstrated that glutamate down-regulates CCTα expression by specifically activating NMDA receptor, then inhibiting PC synthesis in lung tissues. Next, we further studied the mechanism of CCTα expression downregulation at the cellular level in A549 cells. In cell experiments, we directly treated A549 cells with NMDA, an NMDAR-specific agonist. Glutamate can activate other receptors in addition to NMDA receptor, so instead of treating A549 cells with glutamate, we used the specific agonist NMDA. The rescue experiments have been performed using the NMDAR blocker (MK-801) (Figure 7). 4.We have added experiments to illustrate this point. the protein levels of Sp1 in the cytoplasm and nucleus of A549 cells after NMDA treatment were detected by Western blot (Figure 7A, B). As shown in figure 7A-D, The expression of Sp1 is increased in the cytoplasmic proteins and decreased in the nuclear proteins after treated with 300 μM NMDA (P<0.05, P<0.01) (Figure 7C, D). However, 50 μM MK-801 could reverse the increase of Sp1 expression in the cytoplasmic proteins and the decrease in the nuclear proteins induced by NMDA (P<0.05, P<0.01) (Figure 7C, D). 5.In figure 7E, the immunofluorescence staining of Sp1 after NMDA treatment in A549 cells has been re-detected and the Sp1 immunofluorescence data of NMDA treatment has been re-collected. The quality of image data has been improved. We use a magnification of 20×. Higher magnification images are very blurry. 6.In figure 2B, the “Glutamae” has been modified to “Glutamate”. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0289530.r002
22 May 2023 Decision Letter - Yi Cao, Editor PONE-D-22-33593R1Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesisPLOS ONE Dear Dr. Luo, 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: Please carefully address the comments. Please be aware this is the final chance to revise the manuscript============================== Please submit your revised manuscript by Jul 06 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, Yi Cao Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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) ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 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 ****** 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: Although the authors have responded to most of my previous comments and revised the manuscript accordingly, the data quality issues remain. 1. In Figures 4D and 4E, the Western blotting data can not correspond to quantified data. 2. In Figures 7B and 7D, which internal control is used for quantitative analysis? Although β-actin is also present in the nucleus, it is not suitable to be used as a control for nuclear protein samples. 3. In Figure 7E, the image data is highly blurry, which can not support their statement of Sp1 translocation. ****** 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 ******** [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.0289530.r003
Revision 2
23 Jun 2023 Author Response Dear editor and reviewer, Thank you very much for your letter and valuable comments and suggestions for our manuscript. We have modified and improved our manuscript according to your kind advice and the comments raised by the reviewers, and the amendments are highlighted in red in the revised manuscript. We sincerely hope that the revision is acceptable to be published on PLOS ONE. Below, please find the comments in black, followed by our responses in red. Exact changes in the manuscript are also presented in red font. Thank you very much for all your help and looking forward to hearing from you soon. With best wishes, Sincerely yours, First author: Xiao-Hong Li Corresponding authors: Zi-Qiang Luo and Chen Li, Email: luoziqiang@csu.edu.cn and Chen.physiology@outlook.com Reviewer #1: Although the authors have responded to most of my previous comments and revised the manuscript accordingly, the data quality issues remain. Responses: Thank you for your reviews. 1. In Figures 4D and 4E, the Western blotting data can not correspond to quantified data. Response: Although the trend of band changes is not obvious when viewed with the naked eye, after statistical analysis, there are statistical differences between different groups. The analysis data of the bar chart is shown in the tables below. The analysis software we used is Bio Rad's Image Lab software. The bar chart and statistical analysis were performed using Image-Pro Plus 6.0 software. 2. In Figures 7B and 7D, which internal control is used for quantitative analysis? Although β-actin is also present in the nucleus, it is not suitable to be used as a control for nuclear protein samples. Response: We have used the rabbit monoclonal antibody to Lamin A + Lamin C (ab108595), which is the nuclear envelope marker. The strip diagram of LaminA+C is shown in the figure 7C. In figures 7B, the GAPDH is used for quantitative analysis in the cytoplasmic proteins. In figures 7D, the Lamin A+C is used for quantitative analysis in the nuclear proteins. They are reflected in the annotation of the vertical coordinates in Figures 7B and 7D. 3. In Figure 7E, the image data is highly blurry, which can not support their statement of Sp1 translocation. Response: In figure 7E, the image data we uploaded to the submission system is clear. The image data in the PDF of the manuscript is relatively blurry, possibly due to reduced pixels. We suggest that you download the original images in the review system to view it, which may be better. In figure 7E, the green color is Sp1, while the blue color is the nucleus. The green color is Sp1, while the blue color is the nucleus. Compared to the control group, the NMDA treatment group showed a decrease in overlapping green and blue cells, while MK-801 could alleviate the reduction caused by NMDA treatment. This indicates a decrease in Sp1 in the nucleus after NMDA treatment. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0289530.r004
4 Jul 2023 Decision Letter - Yi Cao, Editor PONE-D-22-33593R2Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesisPLOS ONE Dear Dr. Luo, 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 submit your revised manuscript by Aug 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, Yi Cao Academic Editor PLOS ONE Additional Editor Comments: Please carefully address the comments from the reviewer, and inadequate responses to the comments may lead to the rejection [Note: HTML markup is below. Please do not edit.] 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) ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No ****** 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 ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors claim that the NMDA treatment group showed decreased colocalization of Sp1 and nucleus, while MK-801 could alleviate the reduction caused by NMDA treatment. But they choose inappropriate data that may lead readers to confusion and misunderstanding. Because one cell expresses a highly green fluorescence signal, the field intensities lead to saturation in a few milliseconds, resulting in the other cells exhibiting a lower fluorescence signal. The author might explain that the expression of Sp1 was decreased under NMDA treatment, but this figure aims to illustrate the finding of nuclear-cytosol translocation of Sp1. They should provide three images representing blue (DAPI), green (Sp1), or yellow (merge) fluorescent channels, just like in the first version of this article, and quantify the percentage of cells with nuclear and cytoplasmic Sp1. Although the result of nuclear and cytoplasmic protein extraction could support their finding (Effects of NMDA on Sp1 nuclear translocation in A549 cells), the immunofluorescence staining data are not. ****** 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 ******** [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.0289530.r005
Revision 3
12 Jul 2023 Author Response Response: Thank you very much for the valuable feedback from the reviewer. The opinions of the reviewer are very important and reasonable. The Western blot results of nuclear and cytoplasmic protein extraction can totally support our finding that NMDA inhibits Sp1 nuclear translocation in A549 cells. The results of immunofluorescence staining are not very suitable. In order to avoid misleading the readers, we have decided not to present this result in the article. This does not affect our research conclusion (Effects of NMDA on Sp1 nuclear translocation in A549 cells). We have removed the contents about Sp1 fluorescence staining in the article. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0289530.r006
21 Jul 2023 Decision Letter - Yi Cao, Editor Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesis PONE-D-22-33593R3 Dear Dr. Luo, 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, Yi Cao 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 ******** 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 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 ****** 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) ****** 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 ******** https://doi.org/10.1371/journal.pone.0289530.r007
Formally Accepted
31 Jul 2023 Acceptance Letter - Yi Cao, Editor PONE-D-22-33593R3 Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesis Dear Dr. Luo: 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. Yi Cao Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289530.r008

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