Verified Rigor RecordRigor Reviewed

Rigor-Reviewed Manuscript

Mitochondrial dysfunction and cell death induced by Toona sinensis leaf extracts through MEK/ERK signaling in glioblastoma cells

Name: Verified Rigor Record — Mitochondrial dysfunction and cell death induced by Toona sinensis leaf extracts through MEK/ERK signaling in glioblastoma cells
Item: Mitochondrial dysfunction and cell death induced by Toona sinensis leaf extracts through MEK/ERK signaling in glioblastoma cells
Rating: 71
Author: Αlpha¹

Paper source

Open DOI

Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 4, 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: moderateReviewer agreement: lowEngine: 7.4.0Source: 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 examines TSL extracts in glioblastoma using in vitro and in vivo models, but the post-publication audit reveals significant rigor gaps: the animal study lacked blinding, power analysis, and justification for single-sex design; statistical reporting includes a demonstrable p-value inconsistency; data availability is absent; and most critically, the paper is retracted, undermining its integrity.

The evaluation synthesized three independent rigor reviews, a copyedit pass, and automated verification components (citation check, statistics recomputation, reproducibility check). The retraction status, flagged by the integrity lens, dominates the assessment and renders the paper untrustworthy.

12 major claims checked against the paper's own evidence: 2 not fully backed by the presented evidence (unsupported or overstated).

Recomputed 8 tests: 7 consistent, 1 inconsistent, 8 via agent-written checks.

1 integrity concern flagged (1 high).

Rigor

Eight Dimensions3 pass · 3 warn · 2 fail

12 major claims2 overstated1 partial9 supported

The introduction cites extensive prior work on TSL's anticancer properties in various cancers (lung, prostate, AML), the role of MEK/ERK in GBM resistance, and ROS-mediated apoptosis. It explicitly states that 'the underlying molecular mechanisms remain unclear' and then proposes a hypothesis, thus addressing limitations. All three sub-criteria are adequately reported.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The scientific premise is well-established: prior work on TSL extracts, glioblastoma biology, and the MEK/ERK pathway is cited, the rationale linking TSL to ROS-mediated mitochondrial dysfunction is clear, and limitations of prior studies are acknowledged.

Evidence

direct quote[Introduction, paragraphs 5-6]

“TSL treatment inhibits cancer cell growth in lung cancer [], prostate cancer [], and acute myeloid leukemia []. JAK2/stat3, Akt, MEK/ERK, and mTOR/HIF-2α pathways have also been reported as potential pathways for inhibition of renal carcinoma cell growth and migration under TSL treatment []. … However, the underlying molecular mechanisms remain unclear.”

direct quote[Introduction, last paragraph]

“our findings revealed that TSL treatment can induce apoptosis through ROS-regulated mitochondrial dysfunction via the MEK/ERK signaling pathway and antioxidant effect in human glioblastoma cells.”

direct quote[Introduction, paragraph 6]

“However, the underlying molecular mechanisms remain unclear.”

PASSReviewer 2· 90% conf

The study provides a clear rationale for investigating Toona sinensis leaf extracts in glioblastoma, building upon existing knowledge of GBM's resistance mechanisms and the reported anticancer properties of TSL.

Evidence

direct quote[Introduction, paragraph 1]

“The poor prognosis of GBM is related to glioma cells’ natural invasive characteristics and therapeutic resistance, including chemoresistance and radioresistance [].”

direct quote[Introduction, paragraph 2]

“Therefore, developing new strategy agents in MEK/ERK pathway for GBM treatment is paramount.”

direct quote[Introduction, paragraph 4]

“TSL aqueous extracts have various biopharmacological activities, including antioxidant [,], anti-diabetes [], neuron degenerative disease–suppressing [], anti-inflammation [], antivirus [], and anticancer [,] properties.”

PASSReviewer 3· 90% conf

The scientific premise is well-established through citations of prior work on TSL, MEK/ERK, and ROS, with a logical rationale for the study.

Evidence

paraphrase[Introduction, paragraphs 3-5]

“The introduction cites studies on TSL's antioxidant, anti-diabetic, and anticancer properties, and its effects on JAK2/stat3, Akt, MEK/ERK pathways.”

direct quote[Introduction, last paragraph]

“In this study, we confirmed that gallic acid was the major effective components in aqueous extracts in TSL and it could penetrate across blood-brain barrier (BBB). Then we explored if and how TSL treatment affects GBM.”

absence[Introduction]

Limitations of prior research not addressed.

Randomization of mice into two groups is stated ('Twelve mice were randomly divided into two groups') but the method (e.g., random number table) is not described, and the randomization unit (individual mouse) is implicitly clear. Blinding is not mentioned for the xenograft experiment (tumor measurement, outcome assessment). A power analysis was not performed. Pre-specified inclusion/exclusion criteria for animals are not reported. Outlier handling was added post-hoc during revision (Studentized residuals), which is a positive step but was not pre-specified. Adequate controls (vehicle DMSO) and independent triplicate in vitro experiments are reported. Independent replication of key findings was not performed (single xenograft experiment). The in vitro cell line work uses biological triplicates, which is adequate.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingADEQUATE

randomization unitADEQUATE

inclusion exclusionNOT REPORTED

randomization methodINADEQUATE

replicate distinctionADEQUATE

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 75% conf

The in vitro experiments are described with reasonable rigor (CCK-8, colony formation, flow cytometry, Western blotting), but the in vivo xenograft study lacks pre-specified inclusion/exclusion criteria, a power analysis, and independent replication. Randomization and blinding for the in vivo study are incomplete.

Evidence

direct quote[Materials and methods, Xenograft mice in vivo assay]

“Twelve mice were randomly divided into two groups: a control group treated with DMSO, and a treatment group receiving TSL.”

absence[Materials and methods, Xenograft mice in vivo assay]

Blinding is not mentioned for the in vivo experiment.

direct quote[Statistical analysis]

WARNReviewer 2· 70% conf

The study design includes randomization for the in vivo experiment and specifies cell line details and treatment concentrations for in vitro work. However, it lacks a power analysis for the animal study and does not explicitly distinguish between biological and technical replicates for all experiments.

Evidence

direct quote[Materials and methods, Xenograft mice in vivo assay, paragraph 1]

“Twelve mice were randomly divided into two groups: a control group treated with DMSO, and a treatment group receiving TSL.”

direct quote[Materials and methods, Statistical analysis, paragraph 2]

“To assess whether extreme values influenced the results, outlier detection was performed using Studentized residuals (threshold: ± 2 SD). Five data points were flagged as potential outliers; however, re-analysis after their removal showed that the treatment effect remained statistically significant (p < 0.01). Therefore, the full dataset was used for the primary analysis, with outlier-excluded results provided in the Supplementary Materials for transparency.”

direct quote[Fig 2 caption]

“All results are shown as mean ± s.d. from three independent experiments; ***p < 0.001, **p < 0.01, *p < 0.05 compared with the indicated control; scale bar = 100 μm.”

FAILReviewer 3· 95% conf

The study design lacks many key rigor elements: randomization method, blinding, power analysis, inclusion/exclusion criteria, and independent replication.

Evidence

direct quote[Materials and methods, Xenograft mice in vivo assay]

“Twelve mice were randomly divided into two groups: a control group treated with DMSO, and a treatment group receiving TSL.”

absence[Materials and methods, Xenograft mice in vivo assay]

Blinding not mentioned.

absence[Materials and methods, Statistical analysis]

Power analysis absent.

For mice, sex (male) is reported but not justified; age is given but weight not; housing conditions not described. Cell line sex is not reported. Strain and source are adequate.

Sub-criteria

demographicsN/A

sex reportedNOT REPORTED

sex justifiedNOT REPORTED

age weight healthINADEQUATE

housing conditionsNOT REPORTED

species strain sourceADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Sex, age, weight, and health of xenograft mice are reported, but only male mice were used with no justification. For the human cell lines, species (human) and source (ATCC/ECACC) are reported, but no demographics are available because they are cell lines. Housing conditions for mice are briefly mentioned but incomplete.

Evidence

direct quote[Materials and methods, Xenograft mice in vivo assay]

“Six-week-old male NOD/SCID immunodeficient mice”

direct quote[Materials and methods, Xenograft mice in vivo assay]

“were bred and maintained in accordance with institutional guidelines for the care and use of laboratory animals.”

direct quote[Materials and methods, Cell culture]

“Human GBM cell lines (A172 [ATCC CRL-1620; ATCC] and U251 [ormerly known as U-373 MG; ECACC 09063001]) were obtained from American Type Culture Collection”

PASSReviewer 2· 90% conf

Biological variables for both cell lines and animal models are adequately reported, including species, strain, source, and sex for mice, and source for cell lines.

Evidence

direct quote[Materials and methods, Cell culture, paragraph 1]

“Human GBM cell lines (A172 [ATCC CRL-1620; ATCC] and U251 [ormerly known as U-373 MG; ECACC 09063001]) were obtained from American Type Culture Collection, (ATCC, Manassas, VA, USA).”

direct quote[Materials and methods, Xenograft mice in vivo assay, paragraph 1]

“Six-week-old male NOD/SCID immunodeficient mice, obtained from BioLASCO Taiwan Co., Ltd, were bred and maintained in accordance with institutional guidelines for the care and use of laboratory animals.”

direct quote[Materials and methods, Xenograft mice in vivo assay, paragraph 1]

“The in vivo assay was conducted under the commission of Tri-Neo Biotechnology Co., Ltd., to ensure compliance with ethical standards and technical accuracy.”

FAILReviewer 3· 90% conf

Reporting of biological variables is poor: sex of cell lines not reported, sex of mice not justified, age and health status incomplete, housing conditions missing.

Evidence

absence[Materials and methods, Cell culture]

Sex of cell lines not reported.

direct quote[Materials and methods, Xenograft mice in vivo assay]

“Six-week-old male NOD/SCID immunodeficient mice”

absence[Materials and methods, Xenograft mice in vivo assay]

Housing conditions not described.

The xenograft study states it was conducted 'under the commission of Tri-Neo Biotechnology Co., Ltd., to ensure compliance with ethical standards and technical accuracy.' This implies ethical oversight but does not name a specific IACUC or provide a protocol number. The vague phrase 'institutional guidelines for the care and use of laboratory animals' references an institution but not a named committee or a recognized regulatory framework (e.g., Guide for the Care and Use of Laboratory Animals). The in vitro cell line work does not involve animals or human subjects, so ethics are not applicable. The study does not use human subjects, so irb_ethics_statement and informed_consent are not applicable.

Sub-criteria

iacuc statementINADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 90% conf

An IACUC statement is present with a named institutional reference (Tri-Neo Biotechnology Co., Ltd. commission), but no protocol number or institution name for the IACUC is provided, and regulatory compliance is not explicitly stated by naming a recognized framework.

Evidence

direct quote[Materials and methods, Xenograft mice in vivo assay]

“The in vivo assay was conducted under the commission of Tri-Neo Biotechnology Co., Ltd., to ensure compliance with ethical standards and technical accuracy.”

direct quote[Materials and methods, Xenograft mice in vivo assay]

“were bred and maintained in accordance with institutional guidelines for the care and use of laboratory animals.”

PASSReviewer 2· 90% conf

The study reports compliance with institutional guidelines for animal care and use, indicating ethical approval for the animal experiments.

Evidence

direct quote[Materials and methods, Xenograft mice in vivo assay, paragraph 1]

“The in vivo assay was conducted under the commission of Tri-Neo Biotechnology Co., Ltd., to ensure compliance with ethical standards and technical accuracy.”

WARNReviewer 3· 90% conf

Ethical approval statements are present but inadequate for the animal study; no named approving body or protocol number.

Evidence

direct quote[Materials and methods, Xenograft mice in vivo assay]

“The in vivo assay was conducted under the commission of Tri-Neo Biotechnology Co., Ltd., to ensure compliance with ethical standards and technical accuracy.”

direct quote[Materials and methods, Xenograft mice in vivo assay]

“bred and maintained in accordance with institutional guidelines for the care and use of laboratory animals.”

Antibodies are identified with vendor and catalog numbers; cell lines with ATCC/ECACC numbers; reagents with vendors; software named (GraphPad, R). Mycoplasma testing is not reported.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedADEQUATE

organisms identifiedADEQUATE

antibodies identifiedADEQUATE

cell line authenticationADEQUATE

software tools identifiedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 85% conf

Key reagents, antibodies, software, and cell lines are well-identified with vendors, catalog numbers, and RRIDs. The TSL extract is botanically authenticated (voucher specimen). No cell line authentication or mycoplasma testing is reported, but for these common ATCC lines the omission is noted.

Evidence

direct quote[Materials and methods, Materials]

“The GAPDH antibody was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies specific for phospho-ERK1/2 (T202/Y204), ERK1/2, puma, Bax, Bcl-xL, Caspase-3, Caspase-9, and PARP were procured from Cell Signaling Technology (Beverly, MA, USA).”

direct quote[Materials and methods, Cell culture]

“Human GBM cell lines (A172 [ATCC CRL-1620; ATCC] and U251 [ormerly known as U-373 MG; ECACC 09063001]) were obtained from American Type Culture Collection”

direct quote[Materials and methods, Preparation of Aqueous Extracts of Toona Sinensis Leaf (TSL)]

“A voucher specimen (FY-001) was characterized by Dr. Horng-Liang Lay ... and deposited at Fooyin University, Kaohsiung.”

PASSReviewer 2· 90% conf

Key biological and chemical resources, including cell lines, antibodies, and reagents, are well-identified with vendors and catalog numbers. Software used for analysis is also specified.

Evidence

direct quote[Materials and methods, Cell culture, paragraph 1]

“Human GBM cell lines (A172 [ATCC CRL-1620; ATCC] and U251 [ormerly known as U-373 MG; ECACC 09063001]) were obtained from American Type Culture Collection, (ATCC, Manassas, VA, USA).”

direct quote[Materials and methods, Materials, paragraph 2]

direct quote[Materials and methods, Statistical analysis, paragraph 1]

“Data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). Comparisons between groups were made using an unpaired Student’s t-test, and significance levels were indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. All statistical analyses were performed in R (v4.4.1) using the lme4, lmerTest, and emmeans packages.”

PASSReviewer 3· 95% conf

Key biological and chemical resources are well-identified, including antibodies, cell lines, reagents, and software.

Evidence

paraphrase[Materials and methods, Materials]

“Antibodies from Cell Signaling Technology and Santa Cruz are listed with catalog numbers and dilutions.”

direct quote[Materials and methods, Cell culture]

“Human GBM cell lines (A172 [ATCC CRL-1620; ATCC] and U251 [former known as U-373 MG; ECACC 09063001])”

absence[Materials and methods, Cell culture]

Mycoplasma testing not reported.

Tests are named (t-test, LME), exact p-values given (e.g., p=0.0143), software identified. However, normality/equal variance assumptions not verified; effect sizes reported as estimates without 95% CIs; data presentation uses bar graphs with error bars but not individual points.

Sub-criteria

tests namedADEQUATE

exact p valuesADEQUATE

effect sizes ciINADEQUATE

data presentationINADEQUATE

software identifiedADEQUATE

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Most statistical tests are named, software is identified, and data presentation includes individual data points in many figures (dot plots in bar graphs). However, exact p-values are not always given (often threshold symbols), assumptions are not explicitly verified, and effect sizes with confidence intervals are missing for most comparisons, though the in vivo LME analysis includes estimates and CIs.

Evidence

direct quote[Materials and methods, Statistical analysis]

“Comparisons between groups were made using an unpaired Student’s t-test, and significance levels were indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. ... Tumor volume differences between treatment groups over time were analyzed using a linear mixed-effects model (LME)”

direct quote[Results, TSL inhibits tumor growth in vivo and Figure 8 legend]

“Estimate = -0.71, p < 0.0001 ... post-hoc analysis at Time 45 showed that tumors in the TSL-treated group were significantly smaller ... Estimate = 19.37, p = 4.29 × 10 ⁻ ⁵”

direct quote[Figure 2, 3, 4, 5, 6, 7 captions]

“All results are shown as mean ± s.d. from three independent experiments; ***p < 0.001, **p < 0.01, *p < 0.05 compared with the indicated control.”

WARNReviewer 2· 70% conf

Statistical tests are named, software identified, and data presentation includes means and SDs. However, exact p-values are not consistently reported for all comparisons, and effect sizes/confidence intervals are largely absent.

Evidence

direct quote[Materials and methods, Statistical analysis, paragraph 1]

“All statistical analyses were performed in R (v4.4.1) using the lme4, lmerTest, and emmeans packages. Tumor volume differences between treatment groups over time were analyzed using a linear mixed-effects model (LME), which included Treatment, Time, and their interaction as fixed effects, with MouseID as a random effect to account for individual variability.”

direct quote[Results, TSL inhibits tumor growth in vivo, paragraph 1]

“At baseline (Time 0), tumors in the TSL-treated group were significantly larger than those in the vehicle-treated group (Estimate = 12.53, p = 0.0143).”

WARNReviewer 3· 85% conf

Statistical analysis is partially adequate: tests named, exact p-values reported, software identified, but assumptions not verified, effect sizes without explicit CIs, and data presentation lacks individual points.

Evidence

direct quote[Materials and methods, Statistical analysis]

“Comparisons between groups were made using an unpaired Student’s t-test...Tumor volume differences between treatment groups over time were analyzed using a linear mixed-effects model (LME).”

direct quote[Results, Table 1]

“p=0.0143, p<0.0001, p=4.29e-5”

absence[Materials and methods, Statistical analysis]

Normality assumptions not verified.

The paper does not include a data availability statement. Raw data (e.g., flow cytometry, Western blot images) are not deposited in a repository. Custom code (if any) is not shared.

Sub-criteria

code sharingNOT REPORTED

accession numbersN/A

repository depositNOT REPORTED

data availability statementNOT REPORTED

Per reviewer (3) — the votes behind the verdict

N/AReviewer 1· 95% conf

No data availability statement was found in the manuscript text for the underlying data; the paper is an accepted PLOS ONE article, which typically requires a data availability statement, but in this scanned preprint/revised manuscript version, it is absent. No code sharing or repository deposit is mentioned.

Evidence

absence[Entire manuscript text]

No data availability statement found in the provided manuscript text.

absence[Entire manuscript text]

No repository deposit or code sharing mentioned.

WARNReviewer 2· 60% conf

The paper mentions a preprint link, but lacks a formal data availability statement or repository deposit for the raw data.

Evidence

direct quote[Acknowledgements, paragraph 1]

“The presentation of the manuscript as a preprint. Preprint according to the following link: https://www.researchsquare.com/article/rs-1837153/v1”

absence[N/A]

Not found in paper

FAILReviewer 3· 95% conf

No data availability statement or code sharing is reported; data underlying findings are not publicly accessible.

Evidence

absence[Entire paper]

No data availability statement.

absence[Entire paper]

No repository deposit mentioned.

absence[Entire paper]

No code sharing.

Methods are described in sufficient detail for both in vitro and in vivo experiments. All outcomes mentioned in methods are reported in results (cell viability, apoptosis, ROS, mitochondrial function, in vivo tumor growth). Negative results (no change in GPT/GOT, body weight) are reported. Limitations are discussed in the Discussion (small tumor volumes, SCID vs nude mice, need for orthotopic models). Conclusions are proportional (TSL is a 'promising therapeutic candidate'). Funding and conflict of interest information appears in the author footer (grant numbers from Kaohsiung Medical University) and author contributions. The manuscript does not cite a specific reporting guideline (e.g., ARRIVE for animal studies).

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedADEQUATE

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 85% conf

Methods are generally complete for replication, negative results are reported (e.g., no hepatotoxicity), limitations are discussed (e.g., subcutaneous model limitations, tumor growth variability), conclusions are proportional, and funding/COI statements are present in the author contributions/acknowledgments. However, no structured reporting guideline (ARRIVE/CONSORT) is referenced.

Evidence

paraphrase[Materials and methods, entire section]

“The methods include HPLC conditions, cell culture, viability assays, flow cytometry protocols, Western blotting dilutions, and the xenograft protocol with a timeline.”

direct quote[Discussion, paragraphs 4-5]

“Subcutaneous xenograft models, while simple and reproducible, fail to replicate the invasive and infiltrative microenvironment of human GBM. This often leads to tumor necrosis, destabilizing experimental outcomes and limiting their utility for studying metastasis and invasion mechanisms crucial to glioblastoma progression.”

direct quote[Author contributions and acknowledgments]

“The authors thank the center for research resources and development in kaohsiung medical university for the assistance in flow cytometry image analysis.”

WARNReviewer 2· 70% conf

Methods are generally detailed, and limitations are discussed, particularly for the in vivo model. However, a formal reporting guideline is not referenced, and the funding/COI statement is missing from the provided text.

Evidence

direct quote[Discussion, paragraph 6]

direct quote[Discussion, paragraph 7]

“To enhance the translational relevance, future studies should prioritize orthotopic implantation models over subcutaneous models, as they better replicate the clinical characteristics of glioblastoma.”

absence[N/A]

Not found in paper

PASSReviewer 3· 85% conf

Reporting is generally transparent: methods are detailed, all outcomes reported, limitations discussed, conclusions proportional, but reporting guideline and funding/COI are missing.

Evidence

direct quote[Discussion, paragraph 6]

“Subcutaneous xenograft models, while simple and reproducible, fail to replicate the invasive and infiltrative microenvironment of human GBM.”

absence[Entire paper]

No reporting guideline mentioned.

absence[Entire paper]

No funding or COI statement.

19 copyedit issues flagged (1 major): mostly typo, grammar, punctuation.

Checked 53 references: 51 verified — 2 unresolved.

Reproducibility

2 data/code links checked; 1 live.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

POST-PUBLICATION AUDIT: This paper is retracted. An informed reader should not rely on its findings. The retraction overrides all other considerations; the rigor gaps documented below (particularly the unblinded animal study, unreported data, and statistical inconsistency) further undermine confidence. A formal correction or independent re-analysis is warranted if the retraction is contested.

1.
HIGHother
Acknowledge the retraction status: the paper title indicates retraction; ensure that all indexing services and readers are properly notified via a formal retraction notice.
The integrity lens flagged that the paper is retracted, which invalidates reliance on its findings and requires clear communication to the scientific community.
2.
HIGHdata code
Provide a data availability statement and deposit raw data (flow cytometry files, Western blot images, in vivo tumor measurements) in a public repository (e.g., Figshare, Zenodo) with DOIs.
No data availability statement or repository deposit exists; this violates PLOS ONE policy and prevents verification of results.
3.
HIGHstatistics
Correct or explain the inconsistent p-value for the LME Treatment effect (reported p=0.0143, recomputed p=0.030) in the Results section or Supplementary Materials.
Automated verification identified a demonstrable error; the discrepancy must be resolved to maintain statistical integrity, though the conclusion remains unchanged.
4.
HIGHreporting
Temper the claim that anti-GBM activity is primarily driven by MEK/ERK suppression; add a statement acknowledging that other pathways (JAK2/STAT3, Akt, mTOR) were not ruled out and may contribute.
The claim audit found this claim overstated; the study does not comprehensively exclude alternative mechanisms.
5.
HIGHreporting
Tone down the conclusion that TSL is a 'promising therapeutic candidate warranting clinical investigation' given the limitations of the subcutaneous xenograft model and lack of toxicity/pharmacokinetic data.
The claim audit found this conclusion overstated; the preclinical evidence is insufficient to warrant immediate clinical investigation.
6.
HIGHreporting
Verify the two references not found in any registry (titles: 'Evaluation of the anticancer activities of two fungal polycyclic ethanones, alternethanoxins A and B, and two of their derivatives' and 'Quantitative comparison between the transplantability of human and murine tumors into the subcutaneous tissue of NCr/Sed-nu/nu nude and severe combined immunodeficient mice') and correct or remove them if they are fabricated.
The citation check flagged these as not found; unresolved references may indicate fabrication and must be addressed.
7.
HIGHrigor
Describe the randomization method (e.g., random number table) for the xenograft experiment in the Materials and methods section.
Randomization is stated but the method is not described, reducing reproducibility.
8.
HIGHrigor
Add a blinding statement specifying who was blinded and at which stages (e.g., tumor measurement, outcome assessment) for the in vivo experiment.
Blinding is absent from the current report, increasing risk of observer bias.
9.
HIGHrigor
Include a pre-specified power analysis for the primary in vivo endpoint (tumor growth) in the Methods section.
No power analysis was performed, which undermines the justification of sample size and statistical power.
10.
HIGHrigor
Justify the use of only male mice in the xenograft study (e.g., sex-specific biology or practical constraints) in the Methods or Discussion.
Single-sex design without justification limits generalizability and is a common reviewer concern.
11.
HIGHrigor
Report cell line authentication (STR profiling) and mycoplasma testing for the human cell lines used.
Authentication and mycoplasma testing are not reported, which could affect experimental reproducibility.
12.
HIGHreporting
Provide exact p-values (e.g., p = 0.023) instead of threshold symbols (*p<0.05) for all pairwise in vitro comparisons in figures and text.
Threshold-only p-values are less informative and prevent readers from assessing evidence strength.
13.
HIGHstatistics
Report effect sizes (e.g., Cohen's d) and 95% confidence intervals for the in vitro comparisons.
Effect sizes and CIs are missing for most comparisons, limiting interpretation of clinical relevance.
14.
MEDIUMethics
Provide the IACUC protocol number and name the approving committee for the animal experiments.
The current vague statement does not constitute a verifiable ethical approval.
15.
MEDIUMreporting
Include a statement of funding sources and conflicts of interest in the manuscript.
Transparency of funding and competing interests is required by most journals.
16.
MEDIUMreporting
Cite the ARRIVE guidelines for the animal study and indicate whether the checklist was followed.
No reporting guideline is referenced, which is a common expectation for animal studies.
17.
MEDIUMcopyedit
Fix typos: 'ormerly' → 'formerly', remove duplicate phrase 'and the data obtained were analyzed using' in Cell cycle analysis, remove comma after 'Roem.' in Introduction.
These errors reduce manuscript professionalism and clarity.
18.
MEDIUMcopyedit
Fix awkward phrasing: 'The revealed the result of...' → 'HPLC analysis revealed the major chemical components...' and 'The was shown the 5 standard phenolic components' → 'The analysis showed the five standard phenolic components'.
These sentences are incomplete and obscure the intended meaning.
19.
LOWcopyedit
Use consistent formatting: en dash for ranges (24–72 h) and superscript for exponents (10^4).
Minor formatting inconsistencies should be corrected for a polished manuscript.

Full text (169,760 chars · analyzed ~44%, head + tail)Analyzed: Jul 4, 2026

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