Systematic identification of 20S proteasome substrates

For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology.

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Reviewer #3:
Proteasomal degradation is vital in all cells and organisms, and dysfunction or failure of proteasomal degradation is associated with various human diseases, including cancer and neurodegeneration.What is the contribution of the free 20S proteasome over that of the 26S proteasome remains elusive.This is mainly due to the difficulty of separating the function of the 20S proteasome from that of the 26S proteasome.Here, the authors have adapted the limited proteolysis-mass spectrometry (LiP-MS) method to identify 20S proteasome substrates.It is based on the detection of putative 20S proteasome cleavage sites by MS, a method referred to as proteasomal induced proteolysis (PIP)-MS.Using this method, they have identified hundreds of putative 20S proteasome substrates, which are enriched in RNA/DNA-binding proteins that contains intrinsically disordered regions (IDRs), as previously reported in the literature.The novelty comes from the discovery that loss of conformation upon oxidative stress is not the main triggers for 20S-mediated degradation, as the selectivity of 20S proteasomes remains poorly affected by oxidative stress.They further showed that the function of oxidised 20S proteasomes is hampered compared to native 20S proteasome which may explain why cells adapt to oxidative stress by making more proteasomes in a later stage.The catalytic activity of oxidised 20S proteasomes was unaffected by oxidative stress, advocating for an alternative mechanism of proteasome inhibition yet to be discovered.Finally, they found that some 20S proteasome substrates are only partially degraded forming truncated proteins, which may have an altered function.
While it is already known that substrates of the 20S proteasomes are mainly IDR-containing proteins, the work by Pepelnjak and Rogawski et al., does bring significant new advances to the proteasome field: (1) So far there is no methodology to interrogate what are the substrates of free 20S proteasomes in vivo.While the methodology presented by the authors is still in vitro, I think this may be one of the first step toward better understanding substrate specificity of the 20S proteasome.Moreover, PiP-MS can be applied to non-conventional forms of the proteasome such as immunoproteasomes and PA28-capped proteasomes, broadening its use for the scientific community.
(2) Partial protein degradation by the proteasome may be more frequent than previously expected, even if this has not been confirmed by the authors in a more physiological condition.
(3) Oxidised 20S proteasomes have reduced degradative capacity while their catalytic sites remained unaffected, the mechanism yet to be discovered.
Data presented in the manuscript support overall authors' conclusion, although some specific conclusions may need to be strengthened (see below).On the whole, the paper is well written, timely and will bring significant technical advances to the proteasome field.In summary, I do think that this paper will be a good addition to the scientific literature, and I would support publication of this manuscript providing the more specific concerns listed below are addressed.

Major points:
(1) The data presented in Supplementary Fig. 2 are not supporting the authors' conclusion to me: "We observed that these three proteins were degraded in a ubiquitin independent manner in the presence of TAK-243 (Fig. 2E and Supplementary Fig. 2).".No visible decrease of 20S substrates is detected in Supplementary Fig. 2, and it seems even to potentially be higher for CCDC50 and JPT1 at 24h, while the housekeeping gene tubulin remains overall pretty stable.This needs to be clarified.
(2) The authors should add all individual data points on their histograms/curves.
(3) Do the authors have data supporting the conclusion that substrates in Figure 2E are degraded by the 20S proteasome and not just cleaved by other proteases, such as co-treatment with epoxomicin?
(4) This paragraph lacks a bit of clarity to me: "Overall, for 20% of proteins in the human proteome we detected at least three peptide indicative of 20S proteasome cleavage (Fig. 2C).Altered peptides from this set of proteins were either peptides that decreased in intensity (adj.p-value < 0.01, log2(FC) < -1) , completely disappeared or new semi-specific peptides that appeared upon proteasome treatment (Fig. 2C).Overall we identified 280 candidate substrates of the 20S proteasome from 2180 peptides pinpointing proteasome cleaved regions.".It's not clear how the authors have only detected 280 candidates while they have detected at least three peptide indicative of 20S proteasome cleavage for 20% of proteins in the human proteome (~20,000 different proteins in human cells, so ~4000 proteins with peptide indicative of 20S proteasome cleavage?).Do they mean 20% of MS detected proteins or, as stated, 20% of proteins in the human proteome?This needs to be clarified by clearly stating how many peptides they have identified in total and the corresponding number of proteins.From these numbers how many had at least three peptide indicative of 20S proteasome cleavage, and how this going down to 280 candidates at the end.

Minor points:
(1) "Supplementary Fig. 1C" at the end of page 6 should be "Supplementary Fig. 1D".(2) Figure 6A: if "Ctrl/Ox" is referring to lysate, the authors should keep the previous nomenclature "L/L*".What Ctrl, Ox, P and P* mean should also be mentioned in the legend.
(3) Is partial degradation by the 20S proteasome occurring due to the presence of a folded domain in the non-degraded part?This could be analysed or at least discussed.(4) There is a typo end of page 17: "phenomenon, with more than 200 proteins partially processed?by the complex."

Reviewer 1
Pepelnjak et al have taken a very thorough and novel approach to identifying the 20S proteasome substrates with HEK293 cells.Adapting an approach of limited proteolysis (normally using proteinase K) with 20S proteasome was a clever to address a critical question in the proteasome field; what are the substrates of the 20S proteasome?The authors do a lot of great analysis that I believe will be very important for the field at large.This is a high quality paper and I have only clarifying questions/concerns.1) I believe the authors have made a good attempt at describing this approach.It would be very important to improve some of the descriptions of what was actually done.a.How were the MS spectra analyzed to generate the resulting final data set that yields a "peptide sequence with the associated change"?Also, please describe what you mean by "associated change".
We acknowledge the reviewer's comment and recognize the need for additional information concerning the MS spectra analysis.Consequently, we have incorporated a detailed explanation of the PiP-MS analysis, along with the inclusion of two schemes illustrating the process (new Figs.1B-C).To identify 20S proteasome substrates by PiP-MS, peptide abundances for all identified peptides were measured in data-independent mode.A comparison was made between the control and proteasome-treated conditions for all peptides of a given protein.The peptide abundance could either remain unchanged or undergo a significant change between conditions.A peptide was considered significantly changing if a peptide with two protease specific ends (specific peptide) decreased in abundance, if a new peptide with a proteasome cleavage site emerged (semi-specific peptide), or if a specific peptide disappeared entirely upon the addition of the proteasome.To translate peptide-level information into protein-level information, we adhered to a stringent criterion.A protein was considered a substrate if at least 50% of detected peptides showed significant change or when less than 50% showed significant change (processed proteins) but the significantly changing peptides colocalized together in the peptide sequence.
b.In the model, the authors show preservation of a peptide or I guess a new peptide with proteasome treatment.This model is not that clear.It would be nice to see the whole process through for at least one or two example proteins.Draw it out in detail.
As suggested by the reviewer, we have incorporated two schematics (new Figs.1B-C) along with a textual explanation to elucidate the PiP-MS methodology.
c.If a peptide is different in the proteasome treated sample then that protein was deemed a 20S substrate, correct?Again, a beginning to end depiction of the analysis with details would be great.The reviewer is correct there are three situations in which a protein was categorized as a substrate: -The intensity of the peptide exhibited a significant decrease.
-The peptide completely disappeared.
-A new peptide emerged.To elucidate this aspect, we have added a textual explanation accompanied by a schematic description (new Figs.1B-C).
d.Is the "peptide with associated change" the location of where the proteasome would cleave or the peptide that is lost with proteasome?
We agree with the reviewer's observation that in Figure EV1, we did not explicitly indicate whether the observed changes in peptides, following the addition of the 20S proteasome, are attributed to a reduction in intensity, complete disappearance, or emergence of new peptides.To address this, we have made modifications to the figure (now presented as new Fig.EV1C-D) and updated the figure legend accordingly.
e. Why does proteasome primarily cause a decrease in peptides?It seems that with a decrease in one peptide there should be a corresponding increase in a new peptide.I calculated that there are around 150 proteins that show significant decrease or complete loss of associated peptides along with an increase in peptides.Are these not some of the more likely proteasome substrates?These proteins may be the better substrates as they are yielding new peptides that are cleaved by the proteasome.It also may reveal the protein substrates that yield relevant peptides for signaling as suggested by ref. 37.
After the cleavage of a protein region, proteasome-specific peptides are produced.However, these peptides may undergo additional degradation by the proteasome or other cellular peptidases, potentially hindering the detection and analysis of resulting degradation products by MS.Moreover, during degradation, peptides may lose lysine or arginine residues, impeding their ionization and subsequent detection by MS.In our analysis, the minimum peptide length was set to 7 amino acids; therefore, if a peptide has a shorter sequence, it would not be recognized by the search engine.The choice of a 7AA length is based on the fact that shorter peptides are often nonproteolytic and would be automatically excluded from the final analysis.We have now revised the text in the method section accordingly.f.Again, Is the calculated FC based on the control, then is the peptide going down just the one presented.Why would only one peptide of a whole protein being degraded by the proteasome go down.I would expect the best substrates to be the one that have a lot of new peptides that go up and some peptides that go down and some that do not change at all.
Our analysis follows stringent criteria for identifying 20S proteasome substrates.Proteins are classified as substrates only if they have a minimum of 50% of peptides significantly changing or if proteins have significantly changing peptides mapping to the same protein region (for processed proteins with less than 50% changing peptides).We believe that such harsh filtering criteria allowed us to focus on most interesting targets.In the revised version of the manuscript, we provide a clear description of these filtering criteria.
g.The authors should provide the complete Excel data sheets with all the analysis on the sheet so readers can see how the protein substrates were identified using this method.
We agree with the reviewer and have uploaded the complete Excel data sheets containing all relevant information, allowing readers to thoroughly examine the identification of protein substrates using our method.
h.The E1 inhibitor did not seem to lead to an increase in ubiquitination?This seems a bit odd.Please provide some explanation.
We greatly appreciate the reviewer's attention to detail in spotting an error in our work, for which we sincerely apologize.In Supplementary Figure 2, we accidentally presented control gel images that did not include the addition of the E1 ubiquitination inhibitor, TAK-243 to the cellular samples, instead of the intended representation of the treated samples.We have made the necessary corrections and updated the figure, now referred to as EV2, to accurately reflect the outcomes of the TAK-243 treatment.This revised figure clearly illustrates the inhibitory effects on ubiquitination and the 20S proteasome degradation of CCDC50, JPT1, and PCNP.

i. Quantify the aSyn gels.
We thank the reviewer for the suggestion and have made the necessary modification to Figure EV5 (formerly Supplementary Figure 5) by incorporating the quantification data points for the three biological repeats of the α-synuclein gels.

Reviewer #3
Proteasomal degradation is vital in all cells and organisms, and dysfunction or failure of proteasomal degradation is associated with various human diseases, including cancer and neurodegeneration.What is the contribution of the free 20S proteasome over that of the 26S proteasome remains elusive.This is mainly due to the difficulty of separating the function of the 20S proteasome from that of the 26S proteasome.Here, the authors have adapted the limited proteolysis-mass spectrometry (LiP-MS) method to identify 20S proteasome substrates.It is based on the detection of putative 20S proteasome cleavage sites by MS, a method referred to as proteasomal induced proteolysis (PIP)-MS.Using this method, they have identified hundreds of putative 20S proteasome substrates, which are enriched in RNA/DNA-binding proteins that contains intrinsically disordered regions (IDRs), as previously reported in the literature.The novelty comes from the discovery that loss of conformation upon oxidative stress is not the main triggers for 20S-mediated degradation, as the selectivity of 20S proteasomes remains poorly affected by oxidative stress.They further showed that the function of oxidised 20S proteasomes is hampered compared to native 20S proteasome which may explain why cells adapt to oxidative stress by making more proteasomes in a later stage.The catalytic activity of oxidised 20S proteasomes was unaffected by oxidative stress, advocating for an alternative mechanism of proteasome inhibition yet to be discovered.Finally, they found that some 20S proteasome substrates are only partially degraded forming truncated proteins, which may have an altered function.
While it is already known that substrates of the 20S proteasomes are mainly IDR-containing proteins, the work by Pepelnjak and Rogawski et al., does bring significant new advances to the proteasome field: (1) So far there is no methodology to interrogate what are the substrates of free 20S proteasomes in vivo.While the methodology presented by the authors is still in vitro, I think this may be one of the first step toward better understanding substrate specificity of the 20S proteasome.Moreover, PiP-MS can be applied to non-conventional forms of the proteasome such as immunoproteasomes and PA28-capped proteasomes, broadening its use for the scientific community.
(2) Partial protein degradation by the proteasome may be more frequent than previously expected, even if this has not been confirmed by the authors in a more physiological condition.
(3) Oxidised 20S proteasomes have reduced degradative capacity while their catalytic sites remained unaffected, the mechanism yet to be discovered.Data presented in the manuscript support overall authors' conclusion, although some specific conclusions may need to be strengthened (see below).On the whole, the paper is well written, timely and will bring significant technical advances to the proteasome field.In summary, I do think that this paper will be a good addition to the scientific literature, and I would support publication of this manuscript providing the more specific concerns listed below are addressed.
We thank the reviewer for indicating that the study brings significant new advances to the proteasome field.We have carefully studied the reviewers' comments and suggestions, and addressed all of them, as described below.

Major points:
(1) The data presented in Supplementary Fig. 2 are not supporting the authors' conclusion to me: "We observed that these three proteins were degraded in a ubiquitin independent manner in the presence of TAK-243 (Fig. 2E and Supplementary Fig. 2).".No visible decrease of 20S substrates is detected in Supplementary Fig. 2, and it seems even to potentially be higher for CCDC50 and JPT1 at 24h, while the housekeeping gene tubulin remains overall pretty stable.This needs to be clarified.
We greatly appreciate the reviewer's attention to detail in spotting an error in our work, for which we sincerely apologize.In Supplementary Figure 2, we accidentally presented control gel images that did not include the addition of the E1 ubiquitination inhibitor, TAK-243 to the cellular samples, instead of the intended representation of the treated samples.We have made the necessary corrections and updated the figure, now referred to as EV2, to accurately reflect the outcomes of the TAK-243 treatment.This revised figure clearly illustrates the inhibitory effects on ubiquitination and the 20S proteasome degradation of CCDC50, JPT1, and PCNP.
(2) The authors should add all individual data points on their histograms/curves.
We thank the reviewer for this comment and have added where possible the individual data points to the histograms and curves.Please see Figures 2D, 2F, 6B, 7C, EV2D-F, EV3A, EV4B and EV5B.
(3) Do the authors have data supporting the conclusion that substrates in Figure 2E are degraded by the 20S proteasome and not just cleaved by other proteases, such as cotreatment with epoxomicin?
We have previously attempted including epoxomicin in the cycloheximide chase cellular experiments involving the ubiquitination inhibitor TAK-243, however the combination of these three inhibitors causes rapid cell death.Therefore, to validate proteasome mediated degradation and rule out cleavage by other proteases, we preformed time dependent degradation assays in the presence of the 20S proteasome with and without epoxomicin, as shown in Figure 2D.The results indeed indicate that proteins identified as PiP-MS hits are substrates of the 20S proteasome.
(4) This paragraph lacks a bit of clarity to me: "Overall, for 20% of proteins in the human proteome we detected at least three peptide indicative of 20S proteasome cleavage (Fig. 2C).Altered peptides from this set of proteins were either peptides that decreased in intensity (adj.p-value < 0.01, log2(FC) < -1) , completely disappeared or new semi-specific peptides that appeared upon proteasome treatment (Fig. 2C).Overall we identified 280 candidate substrates of the 20S proteasome from 2180 peptides pinpointing proteasome cleaved regions.".It's not clear how the authors have only detected 280 candidates while they have detected at least three peptide indicative of 20S proteasome cleavage for 20% of proteins in the human proteome (~20,000 different proteins in human cells, so ~4000 proteins with peptide indicative of 20S proteasome cleavage?).Do they mean 20% of MS detected proteins or, as stated, 20% of proteins in the human proteome?This needs to be clarified by clearly stating how many peptides they have identified in total and the corresponding number of proteins.From these numbers how many had at least three peptide indicative of 20S proteasome cleavage, and how this going down to 280 candidates at the end.
We acknowledge the reviewer's point regarding the clarity of our phrasing.As a result, we have modified the text to clarify that among the total identified proteins, 20% are categorized as substrates of the 20S proteasome, requiring a minimum of three significantly modified peptides.
We apologize for this oversight and have corrected the text accordingly.
(2) Figure 6A: if "Ctrl/Ox" is referring to lysate, the authors should keep the previous nomenclature "L/L*".What Ctrl, Ox, P and P* mean should also be mentioned in the legend.
We apologize for this mistake and have corrected the Figure accordingly.
(3) Is partial degradation by the 20S proteasome occurring due to the presence of a folded domain in the non-degraded part?This could be analysed or at least discussed.
We thank the reviewer for raising this important point and have added the relevant information to the text indicating that the folded-region acts as a stop signal for 20S proteasome cleavage.
(4) There is a typo end of page 17: "phenomenon, with more than 200 proteins partially processed?by the complex." We thank the reviewer for noticing this typo and have corrected it.Thank you again for sending us your revised manuscript.We think that the performed revisions have satisfactorily addressed the issues raised by the reviewers.As such, I am pleased to inform you that your paper has been accepted for publication.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.
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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.

Laboratory protocol Information included in the manuscript?
In which section is the information available?
(Reagents and Tools In the figure legends: state number of times the experiment was replicated in laboratory.

Yes
The number of experiments was added.
In the figure legends: define whether data describe technical or biological replicates.Yes Information was added.

Ethics
Ethics Information included in the manuscript?
In which section is the information available?
(Reagents and Tools

Not Applicable
No speciments and field samples were used in this study.

Dual Use Research of Concern (DURC)
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools

Reporting
Adherence to community standards Information included in the manuscript?
In which section is the information available?
(Reagents and Tools

Data Availability
Data availability 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?

Yes
PRIDE partner repository and are available via ProteomeXchange with identifier PXD044399 Were human clinical and genomic datasets deposited in a public access-controlled repository in accordance to ethical obligations to the patients and to the applicable consent agreement?

Not Applicable
No human clinical and genomic databases were prepared in this study.
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.
0) -[data type]: [full name of the resource] [accession number/identifier] ([doi or URL or identifiers.org/DATABASE:ACCESSION])-For data quantification: please specify the name of the statistical test used to generate error bars and P values, the number (n) of independent experiments (specify technical or biological replicates) underlying each data point and the test used to calculate p-values in each figure legend.The figure legends should contain a basic description of n, P and the test applied.Graphs must include a description of the bars and the error bars (s.d., s.e.m.).
Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) 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.Not Applicable No human research participants were used in this study.
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.

Sample definition and in-laboratory replication Information included in the manuscript? 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) Table, Materials and Methods, Figures, Data Availability Section) 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.Not Applicable No human participants were used in this study.Studies involving human participants: For publication of patient photos, include a statement confirming that consent to publish was obtained.Not Applicable No human participants were used in this study.Studies involving experimental animals: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Include a statement of compliance with ethical regulations.
Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Not ApplicableNo human participants were used in this study.Studies involving human participants:Studies involving specimen and field samples: State if relevant permits obtained, provide details of authority approving study; if none were required, explain why.

granting approval and reference number for
Table, 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 Our study does not fall under dual use research restrictions.If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable We didn't use agents in this study.If a study is subject to dual use research of concern regulations, is the name of the authority the regulatory approval provided in the manuscript?
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.Not Applicable No tumor marjer prognostic studies were performed in this study.For phase II and III randomized controlled trials, 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.
Table, Materials and Methods, Figures, Data Availability Section)