GSDME-mediated pyroptosis promotes the progression and associated inflammation of atherosclerosis.
The main results reproduced: recomputed values matched the published ones within tolerance.
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
Described-well-enough for R1: 1:1 reproduced (direction + P<0.001) on the paper's own public microarray data GSE43292 via a standard third-party pipeline (GEOquery 2.78.0 + limma 3.66.0). GSDME/DFNA5 (probe 8138602, GPL6244) is significantly higher in atheroma plaque than paired macroscopically intact tissue (32 paired patients): log2FC +0.49, all paired/unpaired Wilcoxon, t-test and limma p-values < 0.001, matching the paper's ***P<0.001 (Fig 3a). Graded within-tol (not exact) because the paper reports only asterisk significance, not the exact test/p-value. Earlier «job» had failed because the shared repro-r-geo env lacked GEOquery and the source build needed system libxml2 (absent on the compute node); fixed by building a bioconda env (geoquery/limma) with CONDA_PKGS_DIRS pointed at the writable «infra» pkgs cache. NOT attempted: R2 scRNA-seq (CeleScope 1.1.7->STAR->Seurat->Monocle on raw Singleron FASTQ PRJNA802316; the hard 20%, Singleron-specific chemistry/whitelist + heavy, documented only) and R3 bulk RNA-seq (analysed on the closed commercial Majorbio Cloud Platform; not reproducible from open tooling). Wet-lab results out of scope. Status partial: the clean public pipeline claim reproduces; the heavier/closed results were not pursued.
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Assessment versions
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v1 current initial assessment Score 85assessed: 2026-06-16 ⛓ 92000923433e
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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
- 2026-06-16
- Rubric version
- not recorded
- Assessed by
- —
- 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 the hypothesis that GSDME-mediated pyroptosis in macrophages aggravates the progression and associated inflammation of atherosclerosis.
- ★ GSDME-mediated pyroptosis promotes atherosclerosis progression and its associated inflammation finding
- ★ GSDME is mainly expressed in macrophages (M1 macrophages) within atherosclerotic plaques finding
- ★ GSDME−/−/ApoE−/− mice show reduced atherosclerotic lesion area and inflammatory response on high-fat diet compared to ApoE−/− controls finding
- ★ ox-LDL induces GSDME expression and pyroptosis in macrophages finding
- ★ Ablation of GSDME in macrophages represses ox-LDL-induced inflammation and macrophage pyroptosis mechanism
- ★ STAT3 directly correlates with and positively regulates GSDME expression mechanism
- ★ GSDME expression is increased in human and mouse atherosclerotic plaques compared to healthy/control tissue finding
- Caspase 3 cleaves GSDME after Asp270 to generate a necrotic N-GSDME fragment that induces pyroptosis mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| single-cell RNA sequencing | human carotid atherosclerotic plaques (endarterectomy) | none | GSDME expression distribution across cell types/clusters | — |
| transmission electron microscopy | human carotid atherosclerotic plaques | none | ultrastructural evidence of macrophage pyroptosis | — |
| immunohistochemistry | human carotid artery plaques vs control (normal abdominal artery) | none | IL-1β, caspase 3, GSDME protein levels (IOD/area) | — |
| Western blot | human carotid atherosclerotic lesions (endarterectomy) | none | GSDME, N-GSDME, cleaved caspase 3 protein levels | — |
| in vitro treatment (Western blot/qPCR implied) | macrophages | ox-LDL | GSDME expression and pyroptosis induction | — |
| Oil red O staining / histology (H&E, MOMA2, Masson trichrome, α-SMA immunostaining) | GSDME−/−/ApoE−/− vs ApoE−/− mice aorta and aortic sinus/brachiocephalic artery | GSDME knockout; high-fat diet 12 wk | atherosclerotic lesion area, necrotic area, macrophage/collagen/SMC content | — |
| quantitative PCR | ApoE−/− mice (ND/HFD) and WT mice aorta | high-fat diet vs normal diet | mRNA of IL-1β, TNF, MCP-1, IL-6, GSDME | — |
| public gene expression dataset analysis (GSE43292) | human atheroma plaques vs macroscopically intact tissue | none | GSDME gene expression | — |
- ▼ Atherosclerotic lesion area reduced in GSDME−/−/ApoE−/− mice compared with ApoE−/− mice on high-fat diet 28%
- ▲ GSDME, caspase 3, and IL-1β protein levels increased in human carotid atherosclerotic plaques vs control vessels P=0.004 (each)
- ▲ GSDME-caspase 3 interaction signal increased in atherosclerotic plaques vs control vessels (proximity ligation assay) P=0.009
- ▲ GSDME expression significantly increased in human atheroma plaques vs intact tissue (GSE43292) P<0.001
- ▲ GSDME and N-GSDME protein/mRNA increased in ApoE−/− mouse aorta after high-fat diet vs normal diet P=0.036 (GSDME protein); P<0.001 (mRNA)
- ▲ Proinflammatory gene mRNA (IL-1β, TNF, MCP-1, IL-6) increased in aorta of high-fat diet ApoE−/− mice vs normal diet/WT P<0.001
- – GSDME predominantly expressed in M1 macrophages among atheroma cell subtypes
- ▼ Necrotic lesion area in brachiocephalic artery decreased in GSDME−/−/ApoE−/− mice vs ApoE−/− mice
- pvalue P=0.004 (IHC GSDME, caspase 3, IL-1β in human atherosclerotic vs control vessels)
- pvalue P=0.009 (in situ proximity ligation assay of GSDME-caspase 3 interaction)
- pvalue P=0.036 (GSDME), P=0.003 (N-GSDME), P=0.303 (cleaved caspase 3) (Western blot of human carotid atherosclerotic lesions)
- pvalue P=0.045 (GSDME protein), P=0.125 (N-GSDME), P<0.001 (GSDME mRNA) (ApoE−/− mouse aorta, western diet vs normal diet)
- pvalue P<0.001 (GSDME expression in human atheroma vs intact tissue (GSE43292))
- fold_change 28% reduction (atherosclerotic lesion area, GSDME−/−/ApoE−/− vs ApoE−/− mice)
- pvalue #P<0.001 (proinflammatory gene mRNA, WD vs ND/WT aortas)
- count 5370 cells (10% mito cutoff); 6758 cells (50% mito cutoff) (scRNA-seq of human carotid atherosclerotic plaques)
Statistical methods review
Model: opusA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study combines human tissue analyses (immunohistochemistry, proximity ligation, Western blot), a public microarray dataset, single-cell RNA-seq of human plaques, and ApoE−/− / GSDME−/− mouse models with in vitro macrophage experiments. Two-group comparisons were made with two-tailed Mann–Whitney U tests (human staining/PLA) and unpaired two-tailed Student's t tests (Western blot, qPCR, GSE43292), while multi-group qPCR comparisons used one-way ANOVA with Bonferroni post hoc. Results were generally reported as mean ± SEM with small per-group n (typically 4–6) and mostly exact P values, except the box-plotted GSE43292 data (median/IQR).
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| two-tailed Mann–Whitney U test | Fig. 1c IHC IOD/area (GSDME, caspase 3, IL-1β); Fig. 1d proximity ligation vs IL-1β | n=6 (Fig. 1c), n=5 (Fig. 1d) | not stated |
| unpaired two-tailed Student's t test | Fig. 3a (GSE43292 GSDME expression), Fig. 3b (Western blot human plaque), Fig. 3c (mouse GSDME/N-GSDME protein), Fig. 3d (mouse GSDME mRNA) | n=32/group (Fig. 3a); n=4/group (Fig. 3b–d) | not stated |
| one-way ANOVA with Bonferroni post hoc | Fig. 3e qPCR of inflammatory genes and GSDME across WD/ND ApoE−/− and WT aortas | n=4/group | not stated |
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Variability for most quantitative comparisons was summarized as mean ± SEM with small per-group n (often 4–6).↳ Could also: Showing SD or a 95% confidence interval, along with individual data points (e.g., scatter/dot plots). — SD and CIs convey the spread and precision of the estimate directly, which is often emphasized for small samples; this would add information about data dispersion alongside the mean.
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Several two-group comparisons (Western blot, qPCR, GSE43292) used the unpaired two-tailed Student's t test.↳ Could also: A nonparametric test (e.g., Mann–Whitney U), or a t test with explicit checks/reporting of normality and equal variance (e.g., Welch's t test). — With small n, normality is hard to confirm; a nonparametric or Welch-based approach makes fewer distributional assumptions and would document how the assumption choice was handled.
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Mann–Whitney U and Student's t tests were applied separately across multiple markers within the same figure (e.g., three proteins in Fig. 1c).↳ Could also: A multiplicity adjustment across the related comparisons (e.g., Benjamini–Hochberg FDR or Holm correction). — Adjusting across a family of related markers controls the overall false-positive rate and would make the multiple-marker testing within one figure explicitly accounted for.
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Group means were the focus of the reported comparisons.↳ Could also: Reporting standardized or absolute effect sizes (e.g., Cohen's d, mean difference with CI) alongside P values. — Effect sizes quantify the magnitude of differences independent of sample size, complementing significance testing and aiding cross-study comparison.
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The public microarray dataset GSE43292 was compared between plaque stages with a two-group t test.↳ Could also: A test matched to the box-plot summary (e.g., Mann–Whitney U) or a moderated approach designed for expression arrays (e.g., limma). — Methods like limma borrow information across genes to stabilize variance estimates and are commonly used for array expression data, and a nonparametric test aligns naturally with median/IQR box-plot reporting.
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Single-cell analyses used clustering, UMAP, pseudotime, GSEA, and GSVA, with mitochondrial-content thresholds of 10% and 50%.↳ Could also: Reporting the specific software packages/versions and parameters, and presenting statistical robustness across the two thresholds. — Documented tool versions, parameters, and sensitivity analyses across cutoffs would make the single-cell pipeline fully reproducible and clarify how threshold choice affects the conclusions.
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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GSDME (and caspase 3, IL1B) protein increased in human atherosclerotic plaques by IHCimaging human carotid artery up 2023×1papers★ This paper is the founder (earliest)
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GSDME knockout reduces atherosclerotic lesion area in ApoE-/- mice (~28%)imaging mouse aorta down 2023×1papers★ This paper is the founder (earliest)
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GSDME gene expression increased in human atheroma plaques (GSE43292)microarray human carotid artery up 2023×1papers★ This paper is the founder (earliest)
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Proinflammatory gene expression (IL1B, TNF, IL6, CCL2) reduced in GSDME-/-/ApoE-/- mouse aortaqPCR mouse aorta down 2023×1papers★ This paper is the founder (earliest)
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Proinflammatory gene expression (IL1B, TNF, IL6, CCL2) increased in ApoE-/- mouse aorta on high-fat dietqPCR mouse aorta up 2023×1papers★ This paper is the founder (earliest)
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GSDME is mainly expressed in M1 macrophages in human plaques (>90% CD68 co-localization)scRNA-seq human carotid artery 2023×1papers★ This paper is the founder (earliest)
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Intact GSDME and cleaved N-GSDME protein increased in human advanced atherosclerotic lesionswestern-blot human carotid artery up 2023×1papers★ This paper is the founder (earliest)
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GSDME protein and mRNA increased in ApoE-/- mouse aorta after high-fat dietwestern-blot mouse aorta up 2023×1papers★ This paper is the founder (earliest)
Citation network
Where this publication sits in the reproducibility-weighted citation graph — what it is built on, and what is built on it. Citation data from OpenAlex.
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Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-36807553
Paper: Wei Y et al. "GSDME-mediated pyroptosis promotes the progression and associated inflammation of atherosclerosis." Nat Commun 2023;14:929. PMID 36807553 · PMCID PMC9938904 · DOI 10.1038/s41467-023-36614-w
Pipeline-derived results in the paper
| # | Reported result | Pipeline / tool | Data | In scope? |
|---|---|---|---|---|
| R1 | GSDME (DFNA5) expression significantly increased in human atheroma plaques vs intact tissue, p<0.001 (Fig 3a) | public microarray re-analysis (group comparison + stats) | GEO GSE43292 (public, 64 samples, GPL6244 Affymetrix Human Gene 1.0 ST) | YES — clear, low-hanging, fully public |
| R2 | scRNA-seq: 5,370 cells, ~11 cell types/clusters; GSDME mainly in M1 macrophage (Fig 2a,b) | CeleScope 1.1.7 → STAR 2.6.1a → Seurat 3.1.2 → Monocle 2.14.0 | SRA PRJNA802316 (raw FASTQ, Singleron platform) | PARTIAL/HARD — the last 20%; raw FASTQ + Singleron-specific chemistry/whitelist + heavy. Documented, not fully attempted. |
| R3 | bulk RNA-seq DE analysis | "Majorbio Cloud Platform" (commercial, closed) + unspecified params | SRA PRJNA802807 | OUT — analysis platform is a closed commercial cloud; not reproducible from described open tooling (docs_insufficient for this result). |
| — | wet-lab (WB, IHC, mouse models, ELISA, flow, knockouts, histology) | manual / experimental | — | OUT — not computational. |
Decision (80/20)
Reproduce R1 as the honest 1:1 data point: GSE43292 is a small, fully public microarray series; the claim (direction + significance of GSDME between the two tissue groups) is precisely pinnable. This is the "third-party tool / public data on the paper's own claim" case the brief explicitly endorses.
R2 is the hard 20% (raw single-cell FASTQ + Singleron-specific CeleScope chemistry, reference build, Seurat clustering to land exactly 5,370 cells / matching cluster identities). Per the brief we do NOT chase it; we document feasibility instead.
R3 out: "Majorbio Cloud Platform" is a closed commercial service — the analysis is not reproducible from the paper's text alone.
Note on metadata mismatch
The auto-enriched code.json lists github.com/singleron-RD/CeleScope and
data.json lists GSE43292. These belong to different results: CeleScope is
the scRNA-seq demux tool (R2, raw data PRJNA802316), while GSE43292 (R1) is an
unrelated public microarray re-analysis that does NOT use CeleScope. We reproduce
R1 (GSE43292) — it is the cleaner, fully-public claim.
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Reproduction footprint
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