Mitochondrial genomic alterations in cholangiocarcinoma cell lines

Athitaya Faipan; Sirinya Sitthirak; Arporn Wangwiwatsin; Nisana Namwat; Poramate Klanrit; Attapol Titapun; Apiwat Jareanrat; Vasin Thanasukarn; Natcha Khuntikeo; Luke Boulter; Hasaya Dokduang; Watcharin Loilome

doi:10.1371/journal.pone.0323844

Peer Review History

Original SubmissionSeptember 23, 2024
23 Sep 2024 Author Response Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. https://doi.org/10.1371/journal.pone.0323844.r001
9 Oct 2024 Decision Letter - Matias Avila, Editor PONE-D-24-41324Mitochondrial genomic alterations in cholangiocarcinoma cell linesPLOS ONE Dear Dr. Loilome, 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. Please submit your revised manuscript by Nov 23 2024 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, Matias A Avila, Ph.D. 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. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 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. [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 ******** 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 manuscript entitled "Mitochondrial genomic alterations in cholangiocarcinoma cell lines" (PONE-D-24-41324) presents an analysis of mtDNA structural variants in cholangiocarcinoma (CCA)-derived cell lines. While the study provides interesting and valuable insights, the current presentation is predominantly descriptive. To enhance the impact and relevance of the findings, additional experimental validation and functional characterization of the identified structural variants are warranted. Major Revisions: 1. To establish the functional significance of the observed mtDNA mutations, the authors should evaluate the expression levels of the genes harboring these mutations through qPCR and confirm protein expression via western blotting. These analyses would provide crucial information on whether the identified mutations result in corresponding changes at the transcriptional or translational levels. 2. The manuscript would benefit from the inclusion of phenotypic characterization of the CCA-derived cell lines. Specifically, conducting colony formation assays and determining the doubling time of each cell line would offer important insights into their proliferative and clonogenic capacities, further contributing to understanding their oncogenic potential. 3. It is also recommended to assess the expression of cholangiocarcinoma-related genes by qPCR, including markers such as CK19, MUC1, MUC5, SOX9, EPCAM, IL6, TGFB1, CXCL10, CD133, UHRF1, and SOX17. This would provide a more comprehensive molecular characterization of the cell lines and allow for better contextualization within the CCA biology landscape. 4. If formalin-fixed, paraffin-embedded tissue samples from the original patient tumors are available (or from another cohort of patients as a validation cohort), performing immunohistochemistry to assess the expression of the corresponding proteins would be highly informative. This would facilitate the correlation of genomic alterations with potential protein-level dysregulation in the tumor tissues. 5. Additionally, should clinical data from the patient donors be accessible, it would be beneficial to include a description of the phenotypic characteristics of each CCA donor sample, such as tumor grade, stage, and clinical outcomes. This would significantly enhance the clinical relevance of the findings. 6. Lastly, the authors could consider performing association analyses between the detected mutations and clinical features of cholangiocarcinoma using publicly available datasets, such as those from The Cancer Genome Atlas (TCGA). Using these data could provide valuable correlations between the mutations identified and key clinical parameters, such as patient survival rates, thereby strengthening the translational impact of the study. ****** 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: Amaya Lopez-Pascual ******** [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.0323844.r002
Revision 1
15 Feb 2025 Author Response RE: PONE-D-24-41324 Dear Editor-in-Chief, We are very thankful again to you and the reviewers for the critical reading of our manuscript entitled “Mitochondrial genomic alterations in cholangiocarcinoma cell lines”, by Faipan et al., to PLOS ONE. We appreciate the reviewers’ comments and suggestions, and have incorporated them into the manuscript as follows: 1. To establish the functional significance of the observed mtDNA mutations, the authors should evaluate the expression levels of the genes harboring these mutations through qPCR and confirm protein expression via western blotting. These analyses would provide crucial information on whether the identified mutations result in corresponding changes at the transcriptional or translational levels. Ans: Thank you for your suggestion. There is the previous study demonstrated that the KKU-213A CCA cell line, which harbors numerous mtDNA SNVs, had ALDH1A3 expression which significantly rises under lactic acidosis (LA) conditions, and correlates with LDHA expression. Higher ALDH1A3 levels have been linked to poorer patient survival, indicating its potential as a prognostic marker, which we have added into the discussion part line 405 to 409 as highlighted in blue (Thamrongwaranggoon et al., 2022). We also assessed the expression of key proteins, including CK19 and IL-6, using Western blotting to investigate potential functional consequences of the observed mtDNA mutations. Our results show that KKU-023, KKU-213A, and KKU-452 exhibit high expression levels of both CK19 and IL-6 proteins, while KKU-055, KKU-100, and MMNK-1 demonstrate lower expression levels (See Response to Reviewer No. 3 below). Moreover, to explore the potential link between mtDNA mutations and protein expression, we analyzed the correlation between CK19 and IL-6 protein expression level and the total number of mtDNA single nucleotide variants (SNVs) and insertions-deletions (INDELs). No significant correlations were found between mtDNA mutations and CK19 protein expression levels, aligning with our literature review, which revealed no prior studies addressing this relationship. In contrast, IL-6 protein expression has been linked to TFB2M, a key regulator of mitochondrial DNA (mtDNA) transcription and maintenance. Overexpression of TFB2M has been shown to increase extracellular mtDNA and IL-6 levels in ovarian cancer cells, promoting M2 macrophage infiltration via the cytoplasmic mtDNA/TLR9/NF-κB/IL-6 pathway (Lei et al., 2024). Although our results did not demonstrate a significant association. We have added this information in the discussion part line 409 to 413 highlighted in blue. 2. The manuscript would benefit from the inclusion of phenotypic characterization of the CCA-derived cell lines. Specifically, conducting colony formation assays and determining the doubling time of each cell line would offer important insights into their proliferative and clonogenic capacities, further contributing to understanding their oncogenic potential. Ans: Thank you for your insightful suggestion. The doubling time and the total number of mtDNA SNVs and INDELs for each cell line have already been provided in the table below. We also analyzed the correlation between them, unfortunately no significant correlation was observed. We put this sentence in the discussion part as in line 415-417 as highlighted in blue. P value of correlation curve between doubling time and total INDEL and total SNV is 0.6741 and 0.4909, respectively. 3. It is also recommended to assess the expression of cholangiocarcinoma-related genes by qPCR, including markers such as CK19, MUC1, MUC5, SOX9, EPCAM, IL6, TGFB1, CXCL10, CD133, UHRF1, and SOX17. This would provide a more comprehensive molecular characterization of the cell lines and allow for better contextualization within the CCA biology landscape. Ans: Thank you for your valuable suggestion. We assessed the expression of CK19 and IL-6 at the protein level using western blot analysis. Our results demonstrate that KKU-023, KKU-213A, and KKU-452 cell lines exhibited high CK19 and IL-6 protein expression, while KKU-055, KKU-100, and MMNK-1 showed low expression levels, as illustrated by the protein intensity bar graphs in Figure 3 below. Additionally, we constructed a correlation curve to evaluate the relationship between CK19 and IL-6 protein intensities and the total number of mtDNA single SNVs and INDELs (Figure 3. And 4.). However, this analysis did not reveal significant correlations.We acknowledge the importance of a more comprehensive molecular characterization and will consider expanding our analysis to include the suggested cholangiocarcinoma-related genes in future studies. 4. If formalin-fixed, paraffin-embedded tissue samples from the original patient tumors are available (or from another cohort of patients as a validation cohort), performing immunohistochemistry to assess the expression of the corresponding proteins would be highly informative. This would facilitate the correlation of genomic alterations with potential protein-level dysregulation in the tumor tissues. Ans: Thank you so much for a great suggestion. We are now conducting the mitochondria mutation analysis in the lager number of cholangiocarcinoma tissues and your suggestion will be absolutely performed. 5. Additionally, should clinical data from the patient donors be accessible, it would be beneficial to include a description of the phenotypic characteristics of each CCA donor sample, such as tumor grade, stage, and clinical outcomes. This would significantly enhance the clinical relevance of the findings. Ans: Thank you for the suggestion. We have added the table of the clinical characteristics of the patient donors from whom the cholangiocarcinoma (CCA) cell lines as in Table 8 of the revised version. 6. Lastly, the authors could consider performing association analyses between the detected mutations and clinical features of cholangiocarcinoma using publicly available datasets, such as those from The Cancer Genome Atlas (TCGA). Using these data could provide valuable correlations between the mutations identified and key clinical parameters, such as patient survival rates, thereby strengthening the translational impact of the study. Ans: We respectfully note that TCGA primarily focuses on nDNA data, including somatic and germline mutations, gene expression, and other features related to nuclear genomics. While TCGA has been invaluable for understanding various aspects of cancer biology, it is not well-suited for the detailed investigation of mtDNA mutations. Unfortunately, the TCGA data does not provide direct or comprehensive coverage of mtDNA as its sequencing methods prioritize nuclear genomes. Although some mtDNA reads can be indirectly extracted from whole-genome or whole-exome sequencing data, these reads are often limited, lack sufficient depth, and can result in incomplete or biased mtDNA mutation profiles. Therefore, conducting meaningful association analyses between mtDNA mutations and clinical features using TCGA data would not be feasible or accurate for this study's specific focus on mitochondrial genomics in CCA. Based on this limitation, we have chosen to rely on direct sequencing and analysis of mtDNA from CCA cell lines in our investigation to ensure robust and accurate identification of mtDNA variants. We believe this approach is more appropriate for achieving the study's aims. We have also added Hasaya Dokduang as the co-author in the revised manuscript because she is the person who handles western blot analysis, which is shown in this response to reviewers’ letter. We believe that the manuscript has been improved satisfactorily and hope that it is now acceptable for publication in PLOS ONE. Yours Sincerely, Watcharin Loilome Attachments Attachment Submitted filename: PONE-D-24-41324R1 Response to reviewers.docx https://doi.org/10.1371/journal.pone.0323844.r003
26 Feb 2025 Decision Letter - Matias Avila, Editor PONE-D-24-41324R1Mitochondrial genomic alterations in cholangiocarcinoma cell linesPLOS ONE Dear Dr. Loilome, 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. Please submit your revised manuscript by Apr 12 2025 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, Matias A Avila, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: 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: Thank you for your detailed responses and thoughtful revisions to the manuscript. I appreciate the effort in incorporating the reviewers' comments and strengthening the discussion. I have a few additional suggestions that would further improve the manuscript: 1. Western Blot Data as Supplementary Material: The Western blot results should be included as supplementary material. Additionally, if not already done, Western blot intensity quantifications should be normalized to β-actin to ensure accurate comparisons. The supplementary material should include the corrected quantification graphs and the corresponding Western blot images. I think it is interesting to have this first molecular characterization. Even if nonsignificant results were found, there are differences in the expression of CK19 specially. the manuscript should mention that further molecular characterization is needed. How many replicates were performed? data are presented as bars with SD or SEM, and is impossible to infer the number of replicates. 2. qPCR Expression Analysis and Correlations: The expression levels of the suggested markers should be evaluated by qPCR. These data should then be used for correlation analyses with doubling time, SNVs, and INDELs. Since qPCR is a straightforward measurement, these additional data would provide valuable support for the characterization, making the findings more robust rather than purely observational. These additions would significantly strengthen the manuscript and provide a more comprehensive molecular characterization of the studied cell lines. I greatly appreciate the inclusion of Table 8 in response to point 5, as it provides valuable information and significantly enhances the clarity of the results. It is a meaningful addition to the manuscript. ****** 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: Yes: Amaya Lopez-Pascual ******** [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.0323844.r004
Revision 2
14 Apr 2025 Author Response 8th April 2025 RE: PONE-D-24-41324R1 Dear Editor-in-Chief, We are very thankful again to you and the reviewers for the critical reading of our manuscript entitled “Mitochondrial genomic alterations in cholangiocarcinoma cell lines”, by Faipan et al., to PLOS ONE. We appreciate the reviewers’ comments and suggestions, and have incorporated them into the manuscript as follows: 1. Western Blot Data as Supplementary Material: The Western blot results should be included as supplementary material. Additionally, if not already done, Western blot intensity quantifications should be normalized to β-actin to ensure accurate comparisons. The supplementary material should include the corrected quantification graphs and the corresponding Western blot images. I think it is interesting to have this first molecular characterization. Even if nonsignificant results were found, there are differences in the expression of CK19 specially. The manuscript should mention that further molecular characterization is needed. How many replicates were performed? data are presented as bars with SD or SEM and is impossible to infer the number of replicates. Author’s response: Thank you for your valuable feedback. We have already performed three independent western blot analysis and intensity normalization to β-actin and included the quantification graphs (Figure S8) along with the corresponding representative western blot images (Figures S7) in the supplementary material. The correlation between IL-6 and CK-19 western blotting intensity with doubling time, SNVs, and INDELs are also provided (Figure S9-10) We also provide the raw data of the three independent western blot analysis as below. 2. qPCR Expression Analysis and Correlations: The expression levels of the suggested markers should be evaluated by qPCR. These data should then be used for correlation analyses with doubling time, SNVs, and INDELs. Since qPCR is a straightforward measurement, these additional data would provide valuable support for the characterization, making the findings more robust rather than purely observational. Author’s response: Thank you for your valuable suggestion. We appreciate your recommendation to include quantitative expression analysis to strengthen our findings. In response, we have performed droplet digital PCR (ddPCR) to quantitatively assess the expression levels of IL-6, CK19, and β-actin across our cell lines. These data are presented in Table S16. Gene expression levels of IL-6 and CK19 were normalized to β-actin for each cell line (Figure S11). Subsequently, we conducted correlation analyses between the normalized gene expression and key cellular features, including doubling time, SNVs, and INDELs. The corresponding correlation plots are shown in Figures S12 and S13. Although the correlation analyses did not yield statistically significant results, likely due to the limited number of cell lines available, we believe the inclusion of these data adds quantitative depth to our study and provides a basis for future investigations with larger sample sizes. S16 Table. Droplet digital PCR results of IL-6, CK-19, and β-actin concentration Sample IL-6 concentration (cp/µL) CK19 concentration (cp/µL) β-actin concentration (cp/µL) KKU-023 33.35 30.52 6666.7 KKU-055 11.82 3.83 6486.3 KKU-100 125.7 7.722 25500 KKU-213A 3785.5 72.38 24500 KKU-452 77.72 0.426 53100 MMNK-1 4582.6 84.21 73000 S12 Fig. Gene expression level of IL-6 after normalized with β-actin (left above) of each cell line demonstrated by droplet digital PCR. Correlation curves of IL-6 gene expression with doubling time (right above), SNVs (left below), and INDELs (right below), respectively. S13 Fig. Gene expression level of CK19 after normalized with β-actin (left above) of each cell line demonstrated by droplet digital PCR (left above). Correlation curves of CK19 gene expression with doubling time (right above), SNVs (middle), and INDELs (below), respectively. We believe that the manuscript has been improved satisfactorily and hope that it is now acceptable for publication in PLOS ONE. Yours Sincerely, Watcharin Loilome, Ph.D. Department of Systems Biosciences and Computational Medicine Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, THAILAND Attachments Attachment Submitted filename: PONE-D-24-41324R2 Response to reviewers.docx https://doi.org/10.1371/journal.pone.0323844.r005
16 Apr 2025 Decision Letter - Matias Avila, Editor Mitochondrial genomic alterations in cholangiocarcinoma cell lines PONE-D-24-41324R2 Dear Dr. Loilome, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Matias A Avila, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0323844.r006
Formally Accepted
Acceptance Letter - Matias Avila, Editor PONE-D-24-41324R2 PLOS ONE Dear Dr. Loilome, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Matias A Avila Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0323844.r007

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 .