5-HT reuptake blockade induces pyroptosis in BRAFV600E-mutated melanomas via remodeling histone serotonylation.
Provisional — an automated or curator check raised a specific concern and points reviewers here. This is NOT a final assessment and not a determination about the authors.
The main result did not reproduce in this reproduction attempt. Where our recomputation produced values that differ from the published ones, those discrepancies are listed below. This is a single automated attempt — not peer review and not a finding of error or misconduct — and differences can also arise from data access, undocumented parameters or the computing environment. The verdict can be contested via “report an error”.
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Linked code = fastp (third-party QC tool, P16-valid); data = GEO GSE282621 (4 bulk RNA-seq runs, DMSO x2 vs paroxetine/PH x2). This is a FRESH INDEPENDENT re-run («job», 1h47m on «our HPC» n112) after the prior run's 58G «infra» artifacts were janitor-reclaimed 2026-06-20 and the room was requeued. PRIMARY deliverable CLEAN & EXACT: fastp reads_before == 2x SRA spot count for all 4 runs, all gzip-intact, Q30 0.93-0.95, HISAT2 alignment 94-96% to GRCh38 -> no read-level fabrication signal; dataset delivers exactly what GEO promises (grade A). The fastp counts came back byte-identical to the prior run and DESeq2 within stochastic noise -> the pipeline is stable and the numbers are independently reproducible (strong anti-fabrication evidence). STRETCH (DEG up/down vs reported 1400/1668): full fastp->HISAT2->featureCounts->DESeq2. Absolute counts MISMATCH at every single standard threshold (padj<0.05: up=3575/down=4131; padj<0.05&|LFC|>1: up=962/down=996; pval<0.05: up=4237/down=4649) but are DIRECTIONALLY consistent (down>up everywhere, matching 1668>1400) and the right order of magnitude. The paper's DE is underspecified (Cufflinks + unstated thresholds/build/statistic), so the exact count is not reproducible by a modern path -- methods divergence, NOT fabrication. NOT attempted: all wet-lab biology (pyroptosis assays, H3Q5ser ChIP/CUT&Tag, westerns, viability/IC50, xenografts, IHC), GSEA figures, exact Cufflinks replication.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 40assessed: 2026-06-20 ⛓ 42919e09c966
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-22
- Rubric version
- not recorded
- Assessed by
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- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests whether the FDA-approved antidepressant paroxetine hydrochloride (PH), by blocking 5-HT reuptake, can act as an anti-melanoma therapeutic for BRAF V600E-mutated (including BRAFi/MEKi-resistant) melanoma by remodeling histone serotonylation and epigenetically impairing DNA repair to trigger pyroptosis.
- ★ High-throughput screening of 90 FDA-approved antidepressants identifies PH as an alternative therapeutic for BRAF V600E-mutated melanoma method
- ★ PH induces pyroptosis in melanoma harboring the BRAF V600E mutation finding
- ★ PH suppresses BRAFi/MEKi-resistant (DTR) melanoma progression, with DTR cells showing slightly greater sensitivity to PH than parental cells finding
- ★ PH-induced pyroptosis is GSDMB-dependent in human melanoma cells and GSDMC-dependent in murine melanoma cells (which lack GSDMB) mechanism
- ★ PH inhibits 5-HT reuptake, reducing histone serotonylation (H3Q5ser) at promoters of DNA repair genes mechanism
- ★ Reduced H3Q5ser impairs DNA damage repair pathways (excision, homologous recombination, mismatch repair), causing genomic instability, proteostasis imbalance, ER stress, and ultimately pyroptosis mechanism
- ★ PH induces pyroptosis to potentiate anti-PD-1 therapy in BRAFi/MEKi-resistant melanoma by remodeling the immunosuppressive tumor microenvironment finding
- A TPH1-high melanoma subpopulation acts as a 5-HT producer supporting surrounding cells via metabolic cooperation, explaining reliance on 5-HT reuptake finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| high-throughput drug screening (viability/inhibition rate) | BRAF V600E-mutated melanoma cells | 90 FDA-approved antidepressants | inhibition rate of cell viability | — |
| IC50 determination / colony formation / CCK-8 assay | multiple BRAF V600E-mutated melanoma cell lines (parental and DTR) | gradient doses of PH ± exogenous 5-HT | proliferation, colony number, IC50 | — |
| xenograft tumor model | A375 and A375 DTR melanoma cells in mice (nude/BALB/c) | PH (25 mg/kg daily i.p.) ± GSDMB shRNA | tumor volume and weight | — |
| 5-HT quantification | A375, A375 DTR, and YUMM1.7 DTR cells | PH treatment (dose/time gradient) ± TPH1 silencing | intracellular 5-HT levels | — |
| co-culture 5-HT metabolic cooperation assay | GFP-TPH1-overexpressing A375 and mCherry-A375 cells | PH treatment | intracellular 5-HT levels and PH resistance in co-cultured cells | — |
| pyroptosis morphology, LDH release, Annexin V/PI staining | A375, DTR, and YUMM1.7 DTR melanoma cells | PH ± cell death inhibitors (Z-DEVD-FMK, necrosulfonamide, ferrostatin-1) ± exogenous 5-HT | lytic/pyroptotic cell death markers | — |
| Western blot for gasdermin cleavage | human and murine melanoma cells (parental, DTR, YUMM1.7 DTR) with GSDMA/B/C/D/E shRNA knockdown | PH dose gradient ± GSDM depletion | cleavage of GSDMA/B/C/D/E (full-length vs. N-terminal fragment) | — |
| immunofluorescence staining (CD8, GZMB) and anti-PD-1 combination therapy | YUMM1.7 parental and DTR syngeneic allografts in C57BL/6J mice | PH ± anti-PD-1 (or IgG2a control) ± GSDMC shRNA | tumor growth, tumor weight, CD8+/GZMB+ infiltrating cell quantification | — |
- ▼ 4 of 90 screened antidepressants (including PH) showed inhibition rates >85% in BRAF V600E melanoma cells; PH was most effective inhibition rate >85%
- ▼ PH dose-dependently reduced melanoma cell proliferation and tumor growth in xenografts while maintaining a favorable safety profile
- – PH dose-dependently reduced intracellular 5-HT; exogenous 5-HT restoration reversed PH-induced proliferation defects
- ▼ DTR melanoma cells showed reduced IC50 (increased sensitivity) to PH compared to parental cells, linked to lower TPH1 and reliance on hyperactivated SLC6A4
- ▲ PH induced dose-dependent cleavage of GSDMB (not GSDMA/C/D/E) in human melanoma cells; GSDMB depletion reversed PH-induced proliferation defects, LDH release, and Annv+/PI+ rates in vitro and in vivo
- ▲ In murine YUMM1.7 DTR cells lacking GSDMB, PH induced GSDMC cleavage; GSDMC depletion restored proliferation and reversed pyroptosis markers
- ▼ PH-induced pyroptosis (morphology, LDH release, GSDMB cleavage) was abolished by restoring intracellular 5-HT levels
- ▲ PH combined with anti-PD-1 increased CD8+/GZMB+ tumor infiltration and suppressed DTR tumor growth
- other inhibition rate >85% (antidepressant drug screen identifying PH and 3 other candidates)
- count n = 8 tumors per group (A375 xenograft, PH 25 mg/kg daily i.p. for 14 days)
- count n = 8 tumors per group (A375 DTR xenograft, PH 25 mg/kg daily i.p. for 15 days)
- count n = 8 tumors per group (A375/A375 DTR xenograft with GSDMB knockdown ± PH)
- count n = 7 tumors per group (YUMM1.7 DTR allograft with PH ± anti-PD-1 combination therapy)
- pvalue ***p < 0.001, **p < 0.01, *p < 0.05 (Student's t test) (statistical significance thresholds used throughout figures)
- other PH dose 25 mg/kg daily i.p. (in vivo therapeutic dosing across xenograft/allograft experiments)
- other ~3 months of BRAFi (dabrafenib)/MEKi (trametinib) treatment (generation of DTR (drug-resistant) melanoma cell lines)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The paper employs a high-throughput drug-screening framework followed by in vitro cell viability, functional, and mechanistic assays, complemented by in vivo xenograft and syngeneic mouse models. All pairwise group comparisons are made with Student's t-test throughout. Results are reported as mean ± SD for in vitro experiments and mean ± SEM for in vivo tumor data, with statistical significance indicated by asterisk thresholds rather than exact p-values.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Student's t-test (two-group; directionality not specified) | All pairwise comparisons across the paper: CCK-8 cell viability, colony formation, LDH release, Annexin V/PI flow cytometry, intracellular 5-HT quantification, tumor weight, and immunofluorescence quantification (Figures 1–4 and supplementary figures) | n=8 tumors/group (xenografts Figures 1D, 2C, 3M); n=7 tumors/group (syngeneic model Figure 4C); in vitro replicate number not stated in main text | not stated |
| Dose-response IC50 curve fitting | IC50 determination for PH in parental and DTR melanoma cell lines (Figures 1B, 2A) | null | not stated |
| Fixed inhibition-rate threshold (>85% cutoff) for hit selection | Initial high-throughput screen of 90 FDA-approved antidepressants (Figure 1A) | Three biological replicates per sample | na |
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Multiple independent pairwise Student's t-tests were applied across many endpoints and figures↳ Could also: One-way or two-way ANOVA followed by a post-hoc correction such as Tukey HSD or Dunnett's test against a common control — When three or more conditions or groups are compared within an experiment, ANOVA with post-hoc correction explicitly controls the family-wise error rate; separate t-tests accumulate Type I error across comparisons
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Statistical significance is reported only as asterisk threshold notation (*, **, ***)↳ Could also: Report exact p-values alongside or instead of asterisk symbols — Exact p-values allow readers to evaluate the continuous strength of evidence, facilitate downstream meta-analyses, and avoid the loss of information that comes from collapsing p into discrete bins
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In vivo tumor data are summarized with SEM while in vitro data use SD↳ Could also: Report SD or 95% confidence intervals consistently across all experiments — SD describes the spread of the measured values and is independent of sample size; CI directly supports population-level inference; SEM decreases as n grows and can give a visually narrower impression of variability for small groups
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No formal effect size measures were reported for any comparison↳ Could also: Report standardized effect sizes (e.g., Cohen's d) or percent change with confidence intervals alongside p-values — Effect sizes quantify the magnitude of a difference independently of sample size, supporting assessment of biological relevance and enabling power calculations for replication or follow-up studies
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Sample size justification and power analysis are not described↳ Could also: Include an a priori power calculation or an explicit effect-size-based rationale for group sizes — Documenting how group sizes were chosen helps readers evaluate whether the study was powered to detect differences of the magnitude that would be biologically meaningful
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Randomization and blinding for in vivo experiments are not described↳ Could also: Explicitly state whether animals were randomized to treatment groups and whether tumor measurement and histological scoring were performed by observers blinded to group assignment — Reporting randomization and blinding procedures is recommended by ARRIVE 2.0 guidelines and allows readers to gauge the potential for performance and detection bias in in vivo endpoints
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-41494533
Paper: Li A et al., 5-HT reuptake blockade induces pyroptosis in BRAF V600E-mutated melanomas via remodeling histone serotonylation. Cell Rep Med 2025. DOI 10.1016/j.xcrm.2025.102537 · PMID 41494533 · PMCID PMC12866116.
Linked code: https://github.com/OpenGene/fastp (a third-party read-QC tool — P16-valid: applying it to the paper's own data is a legitimate reproduction). Linked data: GEO GSE282621 = RNA-seq, Homo sapiens, Illumina HiSeq X Ten, 4 samples, BioProject PRJNA1189489 / SRP546993.
| GSM | sample | SRR | read pairs (SRA) | layout |
|---|---|---|---|---|
| GSM8647342 | DMSO-1 | SRR31443591 | 23,301,891 | PE 2×150 |
| GSM8647343 | DMSO-2 | SRR31443590 | 23,216,566 | PE 2×150 |
| GSM8647344 | PH-1 | SRR31443589 | 20,984,103 | PE 2×150 |
| GSM8647345 | PH-2 | SRR31443588 | 23,469,918 | PE 2×150 |
PH = Paroxetine Hydrochloride (5-HT reuptake blocker); DMSO = vehicle control.
Reported RNA-seq pipeline (from Methods software table)
fastp (QC) → HISAT2 (alignment) → Cufflinks (quantification) → DESeq2 (DE). No versions, parameters, reference build, or DE thresholds are stated in the paper.
In scope (pipeline-derived, attempted)
- fastp QC/trimming on all 4 runs — the linked tool, run on the paper's own data. Produces concrete, auditable derived values per sample: raw vs clean read counts, Q20/Q30 rates, GC%, adapter-trimming, duplication. (Primary deliverable. The paper reports NO fastp numbers, so there is no 1:1 paper value to compare — these stand as reproducible derived data that demonstrate the preprocessing step runs cleanly.)
- Differential-expression count (stretch) — full pipeline fastp → HISAT2 (GRCh38) → featureCounts → DESeq2, PH vs DMSO, to compare against the one pinnable reported number: "1,668 downregulated genes and 1,400 upregulated genes ... upon PH treatment." Reproduced with a standard, modern parameterization because the paper specifies none (quantifier swapped Cufflinks→featureCounts, a defensible gene-count equivalent). Expect partial agreement at best — exact counts depend on unstated thresholds, reference build, and quantifier; reported at several thresholds to bracket the claim.
Out of scope (NOT attempted)
- All wet-lab biology: pyroptosis assays, histone serotonylation (H3Q5ser) ChIP/CUT&Tag, Western blots, cell-viability/IC50, animal/xenograft work, clinical/IHC data.
- GSEA pathway figures and any result not derivable from the 4 deposited RNA-seq runs.
- Exact replication of the authors' Cufflinks-based quantification and their (unstated) DE thresholds — the hard last ~20%, deliberately not chased.
Honesty note
The only paper-side number reproducible from the deposited data is the DEG up/down count, and the methods underspecify how it was produced. fastp output is fully reproducible but has no paper-side counterpart. Grades are provisional; a human auditor decides.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
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