LncRNA DANA1 promotes drought tolerance and histone deacetylation of drought responsive genes in Arabidopsis

Although many long noncoding RNAs have been discovered in plants, little is known about their biological function and mode of action. Here we show that the drought-induced long intergenic noncoding RNA DANA1 interacts with the L1p/L10e family member protein DANA1-INTERACTING PROTEIN 1 (DIP1) in the cell nucleus of Arabidopsis, and both DANA1 and DIP1 promote plant drought resistance. DANA1 and DIP1 increase histone deacetylase HDA9 binding to the CYP707A1 and CYP707A2 loci. DIP1 further interacts with PWWP3, a member of the PEAT complex that associates with HDA9 and has histone deacetylase activity. Mutation of DANA1 enhances CYP707A1 and CYP707A2 acetylation and expression resulting in impaired drought tolerance, in agreement with dip1 and pwwp3 mutant phenotypes. Our results demonstrate that DANA1 is a positive regulator of drought response and that DANA1 works jointly with the novel chromatin-related factor DIP1 on epigenetic reprogramming of the plant transcriptome during the response to drought.

17th Aug 2023 1st Editorial Decision Dear Dr. Wang, Thank you for your patience while your manuscript was peer-reviewed at EMBO reports.We have now received the full set of referee reports as well as referee cross-comments that are all pasted below.As you will see, the referees acknowledge that the findings are potentially interesting.However, they also have several suggestions for how the data could be strengthened.It becomes clear from the cross-comments, that not all requests have to be addressed, for example, the DANA1-ChIPseq is not required.Also some of referee 3's comments do not have to be addressed experimentally.Referee 2 notes that the first point by referee 3 could be addressed, and referee 1 feels that you should address referee 3's points experimentally to the best of your abilities.If you like, we can also discuss the revisions in a video chat.
I would thus like to invite you to revise your manuscript with the understanding that the referee concerns must be fully addressed and their suggestions taken on board.Please address all referee concerns in a complete point-by-point response.Acceptance of the manuscript will depend on a positive outcome of a second round of review.It is EMBO reports policy to allow a single round of major revision only and acceptance or rejection of the manuscript will therefore depend on the completeness of your responses included in the next, final version of the manuscript.
We realize that it is difficult to revise to a specific deadline.In the interest of protecting the conceptual advance provided by the work, we recommend a revision within 3 months (17th Nov 2023).Please discuss the revision progress ahead of this time with the editor if you require more time to complete the revisions.
IMPORTANT NOTE: we perform an initial quality control of all revised manuscripts before re-review.Your manuscript will FAIL this control and the handling will be DELAYED if the following APPLIES: 1) A data availability section providing access to data deposited in public databases is missing.If you have not deposited any data, please add a sentence to the data availability section that explains that.2) Your manuscript contains statistics and error bars based on n=2.Please use scatter blots in these cases.No statistics should be calculated if n=2.
When submitting your revised manuscript, please carefully review the instructions that follow below.Failure to include requested items will delay the evaluation of your revision.1) a .docxformatted version of the manuscript text (including legends for main figures, EV figures and tables).Please make sure that the changes are highlighted to be clearly visible.
3) We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be cited as 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.
-For the figures that you do NOT wish to display as Expanded View figures, they should be bundled together with their legends in a single PDF file called *Appendix*, which should start with a short Table of Content.Appendix figures should be referred to in the main text as: "Appendix Figure S1, Appendix Figure S2" etc. See detailed instructions regarding expanded view here: <https://www.embopress.org/page/journal/14693178/authorguide#expandedview>-Additional Tables/Datasets should be labeled and referred to as Table EV1, Dataset EV1, etc. Legends have to be provided in a separate tab in case of .xlsfiles.Alternatively, the legend can be supplied as a separate text file (README) and zipped together with the Table/Dataset file.4) a .docxformatted letter INCLUDING the reviewers' reports and your detailed point-by-point responses to their comments.As part of the EMBO Press transparent editorial process, the point-by-point response is part of the Review Process File (RPF), which will be published alongside your paper.5) a complete author checklist, which you can download from our author guidelines <https://www.embopress.org/page/journal/14693178/authorguide>.Please insert information in the checklist that is also reflected in the manuscript.The completed author checklist will also be part of the RPF. 6) Please note that all corresponding authors are required to supply an ORCID ID for their name upon submission of a revised manuscript (<https://orcid.org/>).Please find instructions on how to link your ORCID ID to your account in our manuscript tracking system in our Author guidelines <https://www.embopress.org/page/journal/14693178/authorguide#authorshipguidelines>7) Before submitting your revision, primary datasets produced in this study need to be deposited in an appropriate public database (see https://www.embopress.org/page/journal/14693178/authorguide#datadeposition).Please remember to provide a reviewer password if the datasets are not yet public.The accession numbers and database should be listed in a formal "Data Availability" section placed after Materials & Method (see also https://www.embopress.org/page/journal/14693178/authorguide#datadeposition).Please note that the Data Availability Section is restricted to new primary data that are part of this study.* Note -All links should resolve to a page where the data can be accessed.* If your study has not produced novel datasets, please mention this fact in the Data Availability Section.8) At EMBO Press we ask authors to provide source data for the main manuscript figures.Our source data coordinator will contact you to discuss which figure panels we would need source data for and will also provide you with helpful tips on how to upload and organize the files.9) Our journal also encourages inclusion of *data citations in the reference list* to directly cite datasets that were re-used and obtained from public databases.Data citations in the article text are distinct from normal bibliographical citations and should directly link to the database records from which the data can be accessed.In the main text, data citations are formatted as follows: "Data ref: Smith et al, 2001" or "Data ref: NCBI Sequence Read Archive PRJNA342805, 2017".In the Reference list, data citations must be labeled with "[DATASET]".A data reference must provide the database name, accession number/identifiers and a resolvable link to the landing page from which the data can be accessed at the end of the reference.The following points must be specified in each figure legend: -the name of the statistical test used to generate error bars and P values, -the number (n) of independent experiments (please specify technical or biological replicates) underlying each data point, -the nature of the bars and error bars (s.d., s.e.m.), -If the data are obtained from n Program fragment delivered error ``Can't locate object method "less" via package "than" (perhaps you forgot to load "than"?) at //ejpvfs23/sites23b/embor_www/letters/embor_decision_revise_and_review.txt line 56.' 2, use scatter blots showing the individual data points.
Discussion of statistical methodology can be reported in the materials and methods section, but figure legends should contain a basic description of n, P and the test applied.
-Please also include scale bars in all microscopy images.
11) The journal requires a statement specifying whether or not authors have competing interests (defined as all potential or actual interests that could be perceived to influence the presentation or interpretation of an article).In case of competing interests, this must be specified in your disclosure statement.Further information: https://www.embopress.org/competinginterests We would also welcome the submission of cover suggestions, or motifs to be used by our Graphics Illustrator in designing a cover.
As part of the EMBO publication's Transparent Editorial Process, EMBO reports publishes online a Review Process File (RPF) to accompany accepted manuscripts.This File will be published in conjunction with your paper and will include the referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.
You are able to opt out of this by letting the editorial office know (emboreports@embo.org).If you do opt out, the Review Process File link will point to the following statement: "No Review Process File is available with this article, as the authors have chosen not to make the review process public in this case."I look forward to seeing a revised form of your manuscript when it is ready.

Yours sincerely,
Esther Schnapp, PhD Senior Editor EMBO reports Referee #1: The manuscript tilted 'LncRNA DANA1 promotes histone deacetylation of drought responsive genes in Arabidopsis' by Cai, Zhang, He and Jiang et al describes the role of long non coding RNA DANA1 in regulating drought stress response.They showed that it interacts with DIP1 which is part of a protein complex with PWWP3 and HDA9 which eventually regulates the histone enrichment of drought responsive CYP707A1 and CYP707A2 loci.Though the manuscript highlights the emerging role of lncRNAs in drought stress, some key issues need to be addressed.
Major points: 1.It is interesting to study the dana1 mutant as plants with enhanced ABA catabolism.It is obvious (Fig. 5G) that dana1 mutants have reduced ABA levels under control conditions which increase in response to drought, albeit to a lower level.So, it is worthwhile to discuss that dana1 may not be having a drought specific phenotype only but a general ABA deficient phenotype.Is drought tolerance just one of many responses as dana1 mutants might be highly pleiotropic as seen in many epigenetic machinery mutants.In this regard, it will be great to study the DANA1 expression during different plant developmental (pre-and post-germination) stages.dana1 mutants can be tested in detail for their germination, stomatal development and transpiration rate and water use efficiency during seedling and adult plant stages before and after PEG or drought treatment.
2. The good test that DANA1 regulates the CYP707 expression, would be to test the ABA levels in DANA1 OE lines.In that respect it will be interesting to check whether OE lines have a cyp707 mutant like phenotype like Decreased stomatal index and density (Tanaka et al Plant J 2013).
3. Are the drought phenotype in dana1 and dip1 mutants ABA dependent?Can authors comment on what regulates the expression of DANA1 itself during drought treatment.Is there a possibility of a feedback loop regulation.It will be great to test the expression of DANA1 in ABA signaling mutants like pyl/pyr.4. Though authors have nicely tested the histone enrichment in Fig. 5, however, it will be great to test the dynamic histone acetylation before and after the drought treatment around these loci. 5.As DIP is a ribosome machinery interacting protein, does the authors check the effect of DIP mutation on translation of the proteins.Can the drought phenotype in dip mutant be partly because of translation impairments in the mutant.As the ribosomal machinery is localized in the cytoplasm, does the authors check the localization of DIP alone.
Minor Points: 1.The authors should introduce and discuss the ABA catabolism and CYP707 mutants phenotypes in detail.2. Please explain in detail how the PEG plates were made in Methods section.3. Why authors choose 10 d time point for ChIPs while the DANA1 expression peaked at 6 days after drought.4. Can authors comment why there was no ABA related GO category getting enriched in RNA seq data? 5.Even though authors have discussed this, it will be interesting to comment on the parts of DANA1 which are critical for DIP binding (related to Fig. 3) 6.From RNA seq, defense GO is enriched in the mutant.It would be interesting to test them for pathogen stress as well in future.

Referee #2:
This is a manuscript that explores the role of a new lncRNA DANA1 in drought response.The authors have done a good job by exploring the different aspects of the physiological drought response.I find the drought tests, water loss, root elongation and stomatal aperture analysis of good quality and relevant for the manuscript.The analysis of the DANA1 lncRNA is also of good quality and extensive with knockouts, complementation lines, and overexpressors.
The link between DANA1 and DIP1 protein is also well documented.

I have several suggestions that could improve the work further:
The list of proteins identified in the triple hybrid screen should be shown in the supplement.
The link between DANA1 and CYP7007A1/2 is not well explained I am not sure how did the authors select these targets?Also while I do appreciate the CYP data a DANA1 ChIPseq would additionally strengthen the work.
In summary, I find this a solid piece of work.

Referee #3:
In this manuscript, the authors investigated the function of the drought-induced long intergenic noncoding RNA DANA1 in Arabidopsis.It was shown that DANA1 interacts with the L1p/L10e family member protein DANA1 INTERACTING PROTEIN 1 (DIP1) in the cell nucleus of Arabidopsis.Furthermore, DIP1 alters the histone deacetylase HDA9 binding on CYP707A1 and CYP707A2 loci through direct interaction with PWWP3, a member of the PEAT complex acting via histone deacetylation, thereby repressing CYP707A1 and CYP707A2 expression.It was proposed that DANA1 functions a positive regulator of drought response and works jointly with the chromatin-related factor DIP1 to modulate the epigenetic reprogramming of the plant transcriptome during the response to drought.This study provides a new perspective on how noncoding RNAs function in epigenetic regulation of gene expression in plants.However, further evidence is needed to support these findings.
Major points: 1.More genetic analysis is required to investigate the interaction among DANA1, DIP1, HDA9 and PWWP3.The authors need to generate double and/or triple mutants to further analysis their interactions and functions.Furthermore, DANA1 can be overexpressed in dip1/hda9/pwwp3 mutant background or vice versa to analyze their interaction.
2. I suggest that the authors also need to carry RNA-seq of WT and dana1-2 plants under stress conditions, which will provide more information about the function and activity of DANA1 in stress responses.
3. In Figure 4, pull down assays and BiFC assays were used to analyze the interaction between DIP1 and PWWP3.The authors need to further analyze the interaction between PWWP3 and DIP1 by CoIP assays.
4. PWWP3 is known to belong to the PEAT complex that associates with the histone deacetylase HDA9.Dose DIP1 also interact with HDA9?The authors need to analyze this possibility.
Cross-comments from referee 1: I agree that the DANA1 Chipseq is not a mandatory requirement.I think the authors might be able to address some of the comments by referee 3 experimentally.For others, they might find a way to answer them in some way.
Cross-comments from referee 2: I read the reviews and quickly looked at the manuscript to remind myself about it.As for the comments by the other reviewers, I think the Reviewer one has a good point that DANA could show generic ABA misregulation rather than drought-specific phenotypes in that sense it would be nice to address it by checking ABA levels in nonstressed plants or ABA-related phenotypes as described by him or her.As for Reviewer 3 I think point one has merit in making the work more coherent.In that sense that some of the double mutant analyses proposed or ox of DANA in one of the mutants will strengthen the coherence of the work, in my view they are not absolutely necessary but would be nice.I personally think the other points go a bit too far in confirming what is already nicely shown by the authors.
Cross-comments from referee 3: I agree with you that DANA1-ChIPseq data are not required.I think the authors need to address other comments from both reviewers.
Dear Dr. Esther Schnapp, Thank you very much for the comments on the manuscript (EMBOR-2023-57737-T) entitled "LncRNA DANA1 promotes histone deacetylation of drought responsive genes in Arabidopsis" that we submitted to EMBO reports.The paper has now been revised according to the suggestions, and our point-by-point responses to reviewers' concerns are detailed as follows: Rely to Referee #1: 1.It is interesting to study the dana1 mutant as plants with enhanced ABA catabolism.
It is obvious (Fig. 5G) that dana1 mutants have reduced ABA levels under control conditions which increase in response to drought, albeit to a lower level.So, it is worthwhile to discuss that dana1 may not be having a drought specific phenotype only but a general ABA deficient phenotype.Is drought tolerance just one of many responses as dana1 mutants might be highly pleiotropic as seen in many epigenetic machinery mutants.In this regard, it will be great to study the DANA1 expression

Response:
The subcellular localization of the DIP1 was analyzed using UBQ10: DIP1-GFP transgenic lines, and we found that DIP1-GFP protein resides in the nucleus (Appendix Fig S6E and F).Moreover, the observation that the subcellular localization of DIP1 is in the nucleus is also supported by using the DIP1 complementation line (Figure R2).These results together indicate that DIP1 is not a canonical ribosome component, thus we do not check the effect of DIP1 mutation on protein translation in this study.Response: As per the reviewer's advice, sentences regarding ABA catabolism and CYP707 mutant's phenotype have been added into our revised manuscript as "The phytohormone abscisic acid (ABA) is the major signaling molecule in plant responses to drought stress.ABA content increases when a land plant is subjected to drought stress, and it rapidly decreases when the land plant is recovering from drought.ABA content in plants is modulated by the balance between its biosynthesis and catabolism, and ABA is catabolized via two pathways, hydroxylation and glucose conjugation (Cutler & Krochdo, 1999;Nambara & Marion-Poll, 2005).The 8'-hydroxylation is thought to be the predominant pathway for ABA catabolism, which is catalyzed by a cytochrome P450 monooxygenase encoded by CYP707As (Kushiro et al., 2004;Saito et al., 2004)."(Page 4-5 lines 80-88), and please also see the response to question 1 from Reviewer 1.Your manuscript will be processed for publication by EMBO Press.It will be copy edited and you will receive page proofs prior to publication.Please note that you will be contacted by Springer Nature Author Services to complete licensing and payment information.

Appendix
You may qualify for financial assistance for your publication charges -either via a Springer Nature fully open access agreement or an EMBO initiative.Check your eligibility: https://www.embopress.org/page/journal/14693178/authorguide#chargesguideShould you be planning a Press Release on your article, please get in contact with embo_production@springernature.com as early as possible in order to coordinate publication and release dates.
If you have any questions, please do not hesitate to contact the Editorial Office.Thank you for your contribution to EMBO Reports.

EMBO Press Author Checklist USEFUL LINKS FOR COMPLETING THIS FORM
The EMBO Journal -Author Guidelines EMBO Reports -Author Guidelines Molecular Systems Biology -Author Guidelines EMBO Molecular Medicine -Author Guidelines Please note that a copy of this checklist will be published alongside your article.

Abridged guidelines for figures 1. Data
The data shown in figures should satisfy the following conditions: ➡ ➡ definitions of statistical methods and measures: -are tests one-sided or two-sided?-are there adjustments for multiple comparisons?-exact statistical test results, e.g., P values = x but not P values < x; -definition of 'center values' as median or average; -definition of error bars as s.d. or s.e.m.

Materials
Newly Created Materials Information included in the manuscript?
In which section is the information available?
(Reagents and Tools New materials and reagents need to be available; do any restrictions apply?Yes Materials and Methods

Antibodies
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools For antibodies provide the following information: -Commercial antibodies: RRID (if possible) or supplier name, catalogue number and or/clone number -Non-commercial: RRID or citation

DNA and RNA sequences Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Short novel DNA or RNA including primers, probes: provide the sequences.

Yes
Appendix Table S1 Cell materials Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Plants: provide species and strain, ecotype and cultivar where relevant, unique accession number if available, and source (including location for collected wild specimens).

Materials and Methods
Microbes: provide species and strain, unique accession number if available, and source.

Human research participants Information included in the manuscript?
In which section is the information available?
(Reagents and Tools If your work benefited from core facilities, was their service mentioned in the acknowledgments section?Not Applicable

Design
-common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; Please complete ALL of the questions below.Select "Not Applicable" only when the requested information is not relevant for your study.
if n<5, the individual data points from each experiment should be plotted.Any statistical test employed should be justified.Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name).the assay(s) and method(s) used to carry out the reported observations and measurements.an explicit mention of the biological and chemical entity(ies) that are being measured.an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.

Reporting
Adherence to community standards Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Have primary datasets been deposited according to the journal's guidelines (see 'Data Deposition' section) and the respective accession numbers provided in the Data Availability Section?

Materials and Methods
Were human clinical and genomic datasets deposited in a public accesscontrolled repository in accordance to ethical obligations to the patients and to the applicable consent agreement?

Not Applicable
Are computational models that are central and integral to a study available without restrictions in a machine-readable form?Were the relevant accession numbers or links provided?

Not Applicable
If publicly available data were reused, provide the respective data citations in the reference list.

Not Applicable
The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR.
Fig S20A).However, in the plant post-germination stage, the expression of DANA1 Figure S6.Drought-tolerant phenotype of DIP1 over-expressing plants.A, B Detection on the transcript levels (A) and protein levels (B) of DIP1 in DIP1 over-expressing transgenic lines.DIP1 OE-3 and DIP1 OE-5 are Col-0 plants transformed with UBQ10: DIP1-GFP.DIP1 OE-5 has been used in RIP assay presented in Fig. 3D.ACTIN is shown as a loading control.C The DIP1 over-expressing lines are insensitive to PEG treatment.Scale bars = 1 cm.D Root length and fresh weight of seedlings shown in (C).E Immunoblot analyses showing the nucleus and cytoplasmic distributions of DIP1-GFP protein.F Subcellular localization of DIP1-GFP in rosette leaves of 3-week-old DIP1 OE-5 plants.Scale bar = 20 μm.G Drought-tolerance assay.Col-0, DIP1 OE-3 and DIP1 OE-5 plants grown under normal growth conditions for three weeks were subjected to drought stress for eighteen days and then rewatered for three days.Scale bars = 3 cm.H Survival rate after drought treatment (n = 3 biological replates).I Water loss in detached leaves of three-week-old Col-0, DIP1 OE-3 and DIP1 OE-5 plants.Data information: Values shown are means ± SD from three biological replicates.Asterisks represent significant differences determined by Student's t-test (* P < 0.05; ** P < 0.01; *** P < 0.001).

Figure 5 .
Figure 5. H3K9ac and H3K27 marks along the CYP707A1 and CYP707A2 loci are modulated by DANA1 regulation of HDA9 deposition.A Gene structure of CYP707A1 and CYP707A2, indicating exons (boxes) and introns (lines).The locations of the gene regions analyzed by ChIRP-qPCR and ChIP-qPCR are marked.B The presence of H3K9ac was measured on CYP707A1 and CYP707A2 by ChIP-qPCR in WT, dana1-2 and dip1-1 plants.C The presence of H3K27ac was measured on CYP707A1 and CYP707A2 by ChIP-qPCR in WT, dana1-2 and dip1-1 plants.

Figure 4 .
Figure 4. PWWP3 mutants are more sensitive to drought stress pleased to accept your manuscript for publication in the next available issue of EMBO reports.Thank you for your contribution to our journal.

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

Checklist for Life Science Articles (updated January Study protocol Information included in the manuscript? In which section is the information available?
ideally, figure panels should include only measurements that are directly comparable to each other and obtained with the same assay.plotsincludeclearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).a statement of how many times the experiment shown was independently replicated in the laboratory.This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.(ReagentsandTools Table, Materials and Methods, Figures, Data Availability Section)If study protocol has been pre-registered, provide DOI in the manuscript.For clinical trials, provide the trial registration number OR cite DOI.

In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)If sample or data points were omitted from analysis, report if this was due to attrition or intentional exclusion and provide justification.

definition and in-laboratory replication Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)In the figure legends: state number of times the experiment was replicated in laboratory.

In which section is the information available?
Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.For publication of patient photos, include a statement confirming that consent to publish was obtained.
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Not ApplicableStudies involving human participants:

Use Research of Concern (DURC) Information included in the manuscript? In which section is the information available?
(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority

granting approval and reference number for
the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (