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Rigor-Reviewed Manuscript

Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma · 10.18632/oncotarget.11298

Name: Verified Rigor Record — Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma · 10.18632/oncotarget.11298
Item: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma · 10.18632/oncotarget.11298
Rating: 71
Author: Αlpha¹

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Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Interventional Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: highReviewer agreement: moderateEngine: 7.6.0Source: full text

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper presents a clear scientific premise and adequate ethical approvals, but suffers from significant reporting gaps in study design (no randomization/blinding/power analysis), key resources (no cell line authentication/mycoplasma testing), statistical analysis (missing effect sizes and individual data points), and data availability (no repository deposit). The copyedit pass reveals numerous typographical and grammatical errors that undermine clarity.

All eight dimensions were applicable. The three independent reviewers largely converged on the statuses, with minor divergence on data code availability (two 'fail', one 'warn') and reporting transparency (two 'warn', one 'pass'). The statistics verification component checked only 3 tests with full test statistics; all other p-values (threshold-only) were not verified. The citation check identified one retracted reference.

12 major claims checked against the paper's own evidence: 4 not fully backed by the presented evidence (unsupported or overstated).

Recomputed 3 tests: 3 consistent, 0 inconsistent, 3 via agent-written checks.

10 integrity concerns flagged (0 high).

Rigor

Eight Dimensions2 pass · 5 warn · 1 fail

12 major claims1 unsupported3 overstated1 partial7 supported

The introduction cites prior research identifying EF2 as a tumor-associated antigen overexpressed in gastrointestinal, lung adenocarcinoma, ovarian, hepatocellular, and soft-tissue sarcomas (refs 17-22). The rationale is logical: because EF2 is overexpressed in those cancers and involved in cell cycle progression, it may play a role in LSCC. The study addresses the specific gap, stating 'the effect of EF2 on LSCC genesis has yet not been examined and remains unknown'. Limitations of prior work are implicitly acknowledged by noting EF2's role in LSCC is unknown.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedINADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 92% conf

The premise is well-established: prior work on EF2 overexpression in various cancers is cited, and the rationale for studying its role in LSCC is clearly linked to the gap in knowledge.

Evidence

direct quote[Introduction, paragraph 3]

“Recently EF2 is identified as a novel tumor-associated antigen []. It is reported that EF2 has been found to be highly expressed in a variety of malignant tumors, including human gastrointestinal cancers [], lung adenocarcinoma [], ovary cancer [], hepatocellular carcinoma [] and soft-tissue sarcomas [].”

direct quote[Introduction, final paragraph]

“However, the effect of EF2 on LSCC genesis has yet not been examined and remains unknown.”

direct quote[Abstract]

“The present study was designed to uncover the function of EF2 on lung squamous cell carcinoma (LSCC) cancer cell growth and progression.”

PASSReviewer 2· 95% conf

The scientific premise is well-established: prior work on EF2 overexpression in various cancers is cited, the rationale linking EF2 to LSCC development is logical, and the study addresses a knowledge gap (EF2's role in LSCC specifically).

Evidence

direct quote[Introduction, paragraph 3]

direct quote[Introduction, last paragraph]

“However, the effect of EF2 on LSCC genesis has yet not been examined and remains unknown.”

paraphrase[Introduction, last paragraph]

“The paper identifies that EF2's role in LSCC is unknown, but does not discuss methodological limitations of prior studies.”

PASSReviewer 3· 92% conf

The paper establishes a clear scientific premise connecting EF2 overexpression to cancer progression, citing prior work and acknowledging gaps in LSCC research.

Evidence

direct quote[Introduction, paragraph 4]

“However, the effect of EF2 on LSCC genesis has yet not been examined and remains unknown.”

direct quote[Abstract, paragraph 2]

“The present study was designed to uncover the function of EF2 on lung squamous cell carcinoma (LSCC) cancer cell growth and progression.”

paraphrase[Introduction, paragraph 4]

“The paper states that the study was designed to apply 2D-DIGE and MS to identify differential proteins in LSCC, determining the roles of EF2 in carcinogenesis.”

The proteomic comparison uses 7 pairs of LSCC vs. adjacent normal tissues. No randomization or blinding is reported for the tissue selection or cell culture experiments. No a priori power/sample size calculation is provided. Inclusion/exclusion criteria for the 75-case tissue microarray are not stated; only that they were pathologically diagnosed. Outlier handling is not reported. Biological replicate distinction: cell culture experiments note 'All experiments were performed in independent triplicate', but the definition of 'independent' (e.g., separate culture passages) is not specified. Controls: the empty-vector (pcDB) control is adequate; however, no positive control for EF2 overexpression effects is included. Independent replication is not reported.

Sub-criteria

controlsINADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionINADEQUATE

randomization methodNOT REPORTED

replicate distinctionINADEQUATE

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 75% conf

The study design lacks several key rigor elements: no randomization method, no blinding, no power analysis, no pre-specified inclusion/exclusion criteria, and no distinction between biological and technical replicates. However, it does include a positive control (empty vector) and an in vivo component.

Evidence

absence[Materials and Methods, throughout]

No randomization or blinding method is reported for the in vivo or in vitro experiments.

direct quote[Results, 'Overexpression of EF2 enhances tumorigenicity of NCI-H520 cells in vivo']

“To examine whether overexpression of EF2 promoted tumorigenicity in vivo, EF2/pcDNB3.1 and pcDNB3.1 vector-transduced NCI-H520 cells were subcutaneously injected into the left flank of each nude mice and tumor volume was examined daily.”

direct quote[Results, 'The stable overexpression of EF2 affects cell morphology']

“The cells with an empty vector were named NCI-H520/pcDB, and the two transfected cells were called NCI-H520/ pcDB-EF2-1 and NCI-H520/ pcDB-EF2-2.”

WARNReviewer 2· 85% conf

The study has a mixed design (proteomic case-control, cell line experiments, mouse xenograft). Several key design elements are missing: no randomization, blinding, power analysis, pre-specified inclusion/exclusion criteria, or outlier handling. Replicate distinction and controls are partially adequate.

Evidence

absence[Materials and Methods, entire section]

No randomization or blinding is reported for any experiment.

absence[Materials and Methods, 'Statistics' paragraph]

Sample size justification is absent.

direct quote[Materials and Methods, 'Growth curves, colony formation assays and cell cycle analysis by flow cytometry']

“All experiments were performed in independent triplicate.”

WARNReviewer 3· 78% conf

The study design lacks crucial rigor elements: no randomization, blinding, or a priori power analysis. However, inclusion/exclusion criteria, controls, and at least some replication details are present.

Evidence

absence[Methods section]

No randomization method or unit is reported for cell culture or animal experiments.

absence[Methods section]

No blinding of investigators or assessors is reported.

direct quote[Methods, Growth curves, colony formation assays and cell cycle analysis]

“All experiments were performed in independent triplicate.”

paraphrase[Methods, Xenografting nude mice]

“Only states that cells were subcutaneously injected; no randomization or blinding.”

For this preclinical interventional study with human tissue component, all 6 sub-criteria were applicable. Sex_reported: patient sex is given but heavily male (6/1), animal sex is male (nude mice), no justification for single-sex in animal or human specimens. Age_weight_health: human age range and mouse age reported, weight not reported. Species_strain_source: 'nude mice' with source (Dalian Medical University) but no strain designation. Housing_conditions: not reported. Demographics: age, sex reported; race/ethnicity not reported.

Sub-criteria

demographicsINADEQUATE

sex reportedINADEQUATE

sex justifiedNOT REPORTED

age weight healthINADEQUATE

housing conditionsNOT REPORTED

species strain sourceINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Sex and age/health are partially reported but inadequate for the cell line work; species/strain/source and housing conditions are reported only for the xenograft mouse model, not for the cell line. Demographics are reported for human tissue samples.

Evidence

direct quote[Materials and Methods, 'Xenografting nude mice']

“six-week-old male nude mice were purchased and allowed to acclimate one week in the animal facility.”

absence[Materials and Methods, 'Xenografting nude mice']

Housing conditions for mice are not reported.

direct quote[Materials and Methods, 'Specimen collection and proteomic analysis']

“The tissue samples and patient data used in the study were obtained during surgery from LSCC patients (six male and one female) with an age range of 36 to 67 years”

WARNReviewer 2· 80% conf

Key biological variables are partially reported: sex is reported for the clinical cohort (6 male, 1 female), and age range is given, but weight, health status, and housing conditions for mice are absent. Species/strain for mice is not reported; the NCI-H520 cell line is identified with an ATCC number.

Evidence

direct quote[Materials and Methods, 'Specimen collection and proteomic analysis']

“The tissue samples and patient data used in the study were obtained during surgery from LSCC patients (six male and one female) with an age range of 36 to 67 years”

direct quote[Materials and Methods, 'Xenografting nude mice']

“six-week-old male nude mice were purchased and allowed to acclimate one week in the animal facility.”

absence[Materials and Methods, 'Xenografting nude mice']

Housing conditions for mice not described.

WARNReviewer 3· 85% conf

Sex, age, and health status are partially reported, but species/strain/source for mice lacks detail, and housing conditions are absent. Human demographics are reported for the main cohort but sex is biased (6 males, 1 female) with no justification.

Evidence

direct quote[Methods, Specimen collection and proteomic analysis]

“The tissue samples and patient data used in the study were obtained during surgery from LSCC patients (six male and one female)”

absence[Methods, Xenografting nude mice]

Housing conditions for nude mice are not reported.

direct quote[Methods, Ethics statement]

“Mice were obtained from Dalian Medical University Laboratory Animal Center.”

An ethics statement is present: 'All the animal and human experiments were approved by the Medical Ethics Committee of Dalian Medical University.' For human subjects, informed consent is described: 'The patients understood the collecting process, purpose of the specimen, and signed informed consent for specimen collection.' The statement is adequate as it names the approving body. Animal regulatory compliance is implicit through the use of an institutional committee. There is no explicit mention of the Guide for the Care and Use of Laboratory Animals or EU Directive, but the committee approval is sufficient.

Sub-criteria

iacuc statementADEQUATE

informed consentADEQUATE

irb ethics statementADEQUATE

regulatory complianceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The paper reports ethical approval for both human and animal experiments, with a named ethics committee, informed consent for patients, and regulatory compliance for animal care.

Evidence

direct quote[Materials and Methods, 'Ethics statement']

“All the animal and human experiments were approved by the Medical Ethics Committee of Dalian Medical University.”

direct quote[Materials and Methods, 'Ethics statement']

“The patients understood the collecting process, purpose of the specimen, and signed informed consent for specimen collection.”

direct quote[Materials and Methods, 'Ethics statement']

“Mice were obtained from Dalian Medical University Laboratory Animal Center. We anesthetized the mice by CO 2 and then froze them at 4°C.”

PASSReviewer 2· 90% conf

Ethics approval statements are present for both human and animal work: a named committee (Medical Ethics Committee of Dalian Medical University) approved the study, patient informed consent is described, and regulatory compliance is implied via the statement that all experiments were approved.

Evidence

direct quote[Materials and Methods, 'Ethics statement']

“All the animal and human experiments were approved by the Medical Ethics Committee of Dalian Medical University.”

direct quote[Materials and Methods, 'Ethics statement']

“The patients understood the collecting process, purpose of the specimen, and signed informed consent for specimen collection.”

absence[Materials and Methods, 'Ethics statement']

No protocol number or specific regulatory framework cited.

PASSReviewer 3· 90% conf

Ethics approval is clearly stated with a named committee, informed consent was obtained, and regulatory compliance for animal experiments is implied.

Evidence

direct quote[Methods, Ethics statement]

“All the animal and human experiments were approved by the Medical Ethics Committee of Dalian Medical University.”

direct quote[Methods, Ethics statement]

“The patients understood the collecting process, purpose of the specimen, and signed informed consent for specimen collection.”

absence[Methods, Ethics statement]

No explicit reference to animal welfare regulations (e.g., Guide for the Care and Use of Laboratory Animals).

NCI-H520 cells have ATCC number: 'ATCC® HTB182 TM'. The EF2 expression plasmid is described (pcDNA3.1/myc-His(-)B) with primer sequences. Antibodies: primary antibodies for western blotting are mostly identified with catalog numbers (e.g., Akt1 10176-2-AP, ProteinTech; E-cadherin 20874-1-AP; p-Akt antibodies from BioWorld). However, the EF2 monoclonal antibody used for IHC/western blot is from EPITOMICS, China, but no catalog number is given. Secondary antibodies (goat anti-rabbit IgG, Golden Bridge) have only company name. The mouse strain for xenografts is not specified beyond 'nude mice'. No RRIDs are provided. Mycoplasma testing is not reported for NCI-H520 cells.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedADEQUATE

organisms identifiedINADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationINADEQUATE

software tools identifiedADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

Key resources are partially identified: antibodies are reported with vendor and catalog numbers but no RRIDs or clone IDs; the cell line is identified (ATCC number) but not authenticated; organisms are identified only as 'nude mice' without strain; reagents are reported; software is reported with version but no URLs. Several sub-criteria are not applicable.

Evidence

direct quote[Materials and Methods, 'Western blotting analysis']

“Akt1(10176-2-AP, ProteinTech Group; 1: 400), E-cadherin (20874-1-AP, ProteinTech Group; 1:800), β-catenin(17565-1-AP, ProteinTech Group; 1:3000), MMP2(BS1236, BioWorld Technology, Inc., USA; 1:200)”

absence[Materials and Methods, 'Cell culture and stable overexpression of EF2 in NCI-H520 cells']

Cell line authentication (e.g., STR profiling) is not reported.

direct quote[Materials and Methods, 'Xenografting nude mice']

“six-week-old male nude mice were purchased and allowed to acclimate one week in the animal facility.”

WARNReviewer 2· 90% conf

Key biological resources are partially identified: the primary cell line (NCI-H520) has an ATCC number, several antibodies are identified with catalog numbers, and the EF2 plasmid/source is detailed. However, several antibodies lack catalog numbers or RRIDs, and the mouse strain is not specified.

Evidence

direct quote[Materials and Methods, 'Cell culture and stable overexpression of EF2 in NCI-H520 cells']

“The human lung squamous carcinoma NCI-H520 cells (ATCC® HTB182 TM ) were pursued from Cancer Hospital Chinese Academy of Medical Sciences”

direct quote[Materials and Methods, 'Western blotting analysis' and 'Western blotting and LSCC tissue microarrays analysis']

“the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).”

absence[Materials and Methods, 'Cell culture and stable overexpression of EF2 in NCI-H520 cells']

Mycoplasma testing not reported.

WARNReviewer 3· 82% conf

Most reagents and software are identified, but antibodies lack some validation details, cell line authentication is absent, and mycoplasma testing is not reported.

Evidence

absence[Methods, Cell culture and stable overexpression of EF2 in NCI-H520 cells]

Cell line authentication not reported.

absence[Methods, Cell culture and stable overexpression of EF2 in NCI-H520 cells]

Mycoplasma testing not reported.

direct quote[Methods, Western blotting analysis]

“The membranes were probed with antibodies to Akt1(10176-2-AP, ProteinTech Group; 1: 400), E-cadherin (20874-1-AP, ProteinTech Group; 1:800)...”

Tests named: chi-square, Fisher's exact, one-way ANOVA, Student-Newman-Keuls method. Assumptions not verified. Exact p-values: some reported (p=7.5×10^-15, p=0.0008, p=0.054, p=0.081, p=0.403), but for in vitro experiments, only 'p < 0.05' is stated. Effect sizes and confidence intervals not reported. Data presentation: bar charts with error bars (SD stated) are used; individual data points are not shown in all figures; per-group n is stated for some experiments (n=3 triplicate, n=7 pairs for proteomics, n=16 pairs for western blot). Software identified: 'SPSS 13.0 software' for statistical analysis. Mathematical plausibility: no obvious errors in the reported means, Ns, or percentages.

Sub-criteria

tests namedADEQUATE

exact p valuesINADEQUATE

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedADEQUATE

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 75% conf

Statistical tests are named and software is reported, but exact p-values are given only for some comparisons, effect sizes and CIs are absent, data presentation is partially adequate (individual data points not shown), and no assumption verification is reported.

Evidence

direct quote[Materials and Methods, 'Statistics']

“The one-way analysis of variance (ANOVA) test and Student Newman Keuls method were used between the two comparisons among all groups.”

paraphrase[Results, Figures 4,5,6]

“Many figures report only '* p < 0.05' without exact values.”

absence[Results, throughout]

No effect sizes or confidence intervals are reported.

WARNReviewer 2· 85% conf

Statistical tests are named (chi-square, Fisher's exact, one-way ANOVA, Student-Newman-Keuls), but assumptions are not verified, exact p-values are given for some results but not all (e.g., 'p < 0.05' for CCK-8), effect sizes with CIs are absent, and data presentation shows individual points in some figures but not all. No arithmetic errors were detected.

Evidence

direct quote[Materials and Methods, 'Statistics']

“All statistical analyses were performed using SPSS 13.0 software. ... The associations between EF2 expression and clinicopathologic features of LSCC were analyzed using chi-square tests and Fisher's exact test. The one-way analysis of variance (ANOVA) test and Student Newman Keuls method were used”

absence[Results, entire section]

Effect sizes and confidence intervals not reported.

direct quote[Results, 'Stable overexpression of EF2 promotes the proliferation of NCl-H520 cells in vitro']

“The ratio of colony formation of NCI-H520/pcDB-EF2 cells was much higher than that of NCI-H520/pcDB cells (* p < 0.05).”

WARNReviewer 3· 75% conf

Statistical tests are named, exact p-values are reported for key findings, and software is identified. However, assumptions are not verified, effect sizes and CIs are missing, and the data presentation relies heavily on bar graphs without individual points.

Evidence

direct quote[Materials and Methods, Statistics]

“The one-way analysis of variance (ANOVA) test and Student Newman Keuls method were used between the two comparisons among all groups.”

direct quote[Results, EF2 is highly expressed in LSCC tissues]

“p =7.5×10 -15”

absence[Figures]

No individual data points shown in figures; bar graphs with SD only.

There is no data availability statement anywhere in the manuscript. The proteomic data (2D-DIGE and MS) are not deposited in a public repository such as PRIDE or MassIVE. The cell line and plasmid are described but no accession numbers for the constructs or deposition in a repository. Custom code is not mentioned; the analysis software (DeCyder, Mascot, SPSS, ModFit) are commercial and not custom, so code sharing is not applicable. The absence of any data availability statement is a clear reporting gap.

Sub-criteria

code sharingN/A

accession numbersNOT REPORTED

repository depositNOT REPORTED

data availability statementNOT REPORTED

Per reviewer (3) — the votes behind the verdict

N/AReviewer 1· 90% conf

The paper is from 2016 and does not include a data availability statement or code sharing. No public repository deposit or accession numbers are provided for the proteomics data.

Evidence

absence[Whole paper]

No data availability statement is present.

absence[Whole paper]

No repository deposit is mentioned for the proteomics or other data.

paraphrase[Materials and Methods, 'Statistics']

“Only commercial software (DeCyder, SPSS) is used.”

FAILReviewer 2· 95% conf

No data availability statement is present; no raw data or code are deposited. The paper does not mention any repository or accession numbers for the proteomics data (mass spectrometry data), and custom code is not shared.

Evidence

absence[Entire paper, including end matter]

No data availability statement.

absence[Materials and Methods, 'Specimen collection and proteomic analysis']

No repository deposit for proteomics data.

absence[Whole paper]

No accession numbers for genomic or proteomic data.

WARNReviewer 3· 80% conf

No data availability statement is provided, and raw data are not deposited. A plasmid sequence is provided (BC126259) and primer sequences are given, but no code repository.

Evidence

absence[Full text]

No data availability statement found anywhere in the manuscript.

absence[Full text]

Proteomics data (2D-DIGE/MS) not deposited in a public repository.

absence[Full text]

No custom code repository is mentioned.

Methods are fairly detailed: cell culture, transfection, western blot, IHC, qPCR, xenograft procedures are described. Trial registration is not applicable (not a clinical trial). No reporting guideline (e.g., ARRIVE for animal studies) is mentioned. All outcomes listed (proliferation, cell cycle, migration, invasion, EMT markers) are reported, but negative findings (e.g., no effect on G1/S phase distribution) are only noted as 'no significant difference' without exact p-values. Limitations are discussed in the Discussion: 'But further investigations are needed to confirm whether EF2 is a decisive gene for promoting EMT in tumor cells.' and 'the mechanisms of EF2 upregulation in LSCC are not clear.' Conclusions are proportional: the paper states EF2 is 'highly expressed in LSCC' and may 'promote LSCC cell growth, migration and invasion' without overgeneralizing to clinical therapy. Funding sources and COI are disclosed.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedINADEQUATE

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 85% conf

The methods are detailed enough for replication, all outcomes reported, limitations discussed, conclusions proportional, and funding/COI provided. Trial registration and reporting guidelines are not applicable for this preclinical study.

Evidence

direct quote[Footnotes, 'GRANT SUPPORT' and 'CONFLICTS OF INTEREST']

“The authors have declared no conflict of interest. This study was supported by grants from National Basic Research Program of China (973 Program, No. 2012CB967003 to S. Shao)...”

direct quote[Discussion, paragraph 7]

“But further investigations are needed to confirm whether EF2 is a decisive gene for promoting EMT in tumor cells.”

paraphrase[Materials and Methods, all sections]

“Proteomics, cell culture, transfection, CCK-8, flow cytometry, scratch wound, transwell, qPCR, western blotting, IHC, and xenograft procedures are all described in sufficient detail.”

WARNReviewer 2· 85% conf

Methods are described in reasonable detail for replication, but there is no trial registration, no explicit reporting guideline adherence (ARRIVE, CONSORT), and not all outcomes are reported transparently (negative/null results for some EMT markers are only partially reported). Limitations are discussed. Conclusions are generally proportional to the evidence.

Evidence

absence[Materials and Methods]

No reporting guideline adherence mentioned.

direct quote[Results, 'The stable overexpression of EF2 promotes the proliferation of NCl-H520 cells in vitro']

“the percentages of cells in the G1 and S phase showed no significant difference between empty vector-transfected cells and EF2-transfected cells”

direct quote[End matter, 'CONFLICTS OF INTEREST' and 'GRANT SUPPORT']

“CONFLICTS OF INTEREST The authors have declared no conflict of interest. GRANT SUPPORT This study was supported by grants from National Basic Research Program of China (973 Program, No. 2012CB967003 to S. Shao)...”

WARNReviewer 3· 80% conf

Methods are detailed enough for replication, limitations are discussed, conclusions are proportional, and funding/COI are provided. However, no reporting guideline is referenced, and trial registration is not applicable but not mentioned.

Evidence

direct quote[Materials and Methods]

“The expression vector for EF2 was constructed by inserting CDS sequence (2577bp, Nucleotide of EF2 : BC126259)... into the XhoI/ HindIII sites in pcDNA3.1/myc-His(-)B vector.”

absence[Full text]

No reporting guideline (ARRIVE, CONSORT) cited.

direct quote[Discussion, paragraph 2]

“But the mechanisms of EF2 upregulation in LSCC are not clear.”

30 copyedit issues flagged: mostly typo, grammar, consistency.

Checked 38 references: 34 verified — 1 retracted, 3 not checked.

Reproducibility

1 data/code link checked; 1 live.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

This published paper has moderate robustness. An informed reader should weigh the absence of randomization, blinding, and power analysis in the xenograft experiment, the lack of cell line authentication and mycoplasma testing, and the missing data availability statement. The retracted reference (PLoS ONE 2014) should be noted. The overstated claims about clinical utility and early diagnosis warrant caution. A correction or independent re-analysis would be needed to address the most consequential gaps.

1.
HIGHdata code
Add a data availability statement and deposit the proteomics data (2D-DIGE/MS) in a public repository (e.g., PRIDE, ProteomeXchange) with an accession number.
The absence of any data availability statement is a major reporting gap that undermines reproducibility and is a common reason for editorial return.
2.
HIGHother
Remove or replace the retracted reference: 'Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS' (PLoS ONE 2014, doi:10.1371/journal.pone.0095121).
Citing a retracted paper is a serious integrity issue that must be corrected.
3.
HIGHreporting
Provide quantitative data (means, SD, p-values) for the xenograft tumor growth experiment, including tumor volume measurements and statistical comparison between groups.
The claim that EF2 enhances tumorigenicity in vivo is unsupported without numerical data and statistical analysis.
4.
HIGHreporting
Temper the claim 'EF2-targeted therapy could become a good strategy for clinical treatment of LSCC' to reflect that no therapeutic intervention was tested; rephrase as a speculation or future direction.
The claim goes beyond the evidence (in vitro and xenograft data only) and is overstated.
5.
HIGHreporting
Remove or temper the claim 'EF2 has certain clinical values for early diagnosis of LSCC' as no diagnostic performance metrics (sensitivity, specificity, ROC) were provided.
The claim is not supported by the data presented.
6.
HIGHrigor
Report randomization method (e.g., random number table) and blinding for the xenograft experiment and cell-based assays in the Methods section.
Lack of randomization and blinding is a major source of bias in preclinical studies.
7.
HIGHrigor
Include an a priori power analysis for the sample sizes of the animal experiment and tissue microarray, citing the expected effect size and alpha level.
Without power analysis, the sample size justification is absent, raising concerns about the study's ability to detect true effects.
8.
HIGHrigor
Distinguish biological from technical replicates in the colony formation, scratch wound, and transwell assays (e.g., 'n=3 independent biological replicates, each with 3 technical replicates').
The current phrase 'independent triplicate' is ambiguous and prevents assessment of the true variability.
9.
HIGHrigor
Report cell line authentication (e.g., STR profiling) and mycoplasma testing status for NCI-H520 cells in the Methods section.
Without authentication and mycoplasma testing, the identity and health of the cell line are unverified, which can invalidate all cell-based results.
10.
HIGHrigor
Specify the exact strain of nude mice (e.g., BALB/c nu/nu) and provide housing conditions (light cycle, temperature, enrichment) in the 'Xenografting nude mice' section.
Incomplete reporting of animal characteristics and housing conditions limits reproducibility.
11.
HIGHstatistics
Report exact p-values for all comparisons (replace '* p<0.05' with specific values like 'p=0.023') and include effect sizes (Cohen's d) or 95% CI for key comparisons.
Threshold-only p-values are insufficient for readers to assess the strength of evidence.
12.
HIGHstatistics
State whether ANOVA assumptions (normality via Shapiro-Wilk, homogeneity of variance via Levene's test) were checked and report any corrections (e.g., Welch ANOVA) if violated.
Without assumption verification, the validity of the ANOVA results is uncertain.
13.
MEDIUMcopyedit
Correct the typo 'NCl-H520' to 'NCI-H520' throughout the manuscript (Results, Figure legends).
Inconsistent cell line naming is confusing and unprofessional.
14.
MEDIUMcopyedit
Correct the typo 'overexppressed' to 'overexpressed' in the Introduction.
Spelling errors undermine the paper's credibility.
15.
MEDIUMcopyedit
Clarify the sacrifice method for mice: the Ethics statement says 'froze them at 4°C' but the Xenograft section says 'sacrificed by CO2'; reconcile these descriptions.
Inconsistent descriptions of animal sacrifice raise concerns about ethical reporting.
16.
MEDIUMreporting
Provide individual data points (e.g., dot plots) overlaid on bar graphs in all figures to display the distribution and variability of replicates.
Bar graphs alone can hide the underlying data distribution and potential outliers.
17.
MEDIUMreporting
Mention adherence to a relevant reporting guideline, such as ARRIVE for the animal xenograft experiments, in the Methods section.
Referencing reporting guidelines improves transparency and reproducibility.
18.
LOWcopyedit
Correct the typo 'growed' to 'grew' in the Results section.
Grammatical error in a key result statement.
19.
LOWcopyedit
Add a space before parentheses in antibody listings (e.g., 'β-catenin (17565-1-AP)') for consistency.
Minor formatting issue that improves readability.

Full text (45,169 chars)Analyzed: Jul 6, 2026

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