The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2.
The main results reproduced, with only marginal, non-material deviations.
- Nothing in this column.
- 🟡Could not use the authors’ exact input data
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
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 to attempt; outcome = PARTIAL with one notable non-reproduction. RNA-seq pipeline (the headline molecular axis) reproduced cleanly from the authors' own GEO count matrix (GSE196328, 10 samples): DESeq2 1.50.2 at FDR<0.05, CPM filter -> 14070 genes, both genotype and differentiation contrasts run. The KO POSITIVE CONTROL is clean (Cdkn1b/p27 strongly DOWN in p27KO, padj 2.7e-11), validating sample labels + pipeline. HOWEVER the headline transcriptomic claim C3 -- 'Sox2 de-repressed (UP) in p27KO' -- does NOT reproduce at the bulk-mRNA level (Sox2 log2FC slightly negative, never significant). This is flagged for human review: the paper most likely demonstrates Sox2 de-repression via protein/ChIP/reporter (wet-lab, out of scope) and bulk neurosphere RNA-seq can dilute cell-type-specific effects -- NOT asserted as fabrication. ATAC-seq (C5/C6) is NOT reproducible because GEO GSE196285 deposits only the 10 MACS2 narrowPeak BEDs (no FASTQ/BAM/bigwig): the Bowtie2/MACS2/DiffBind pipeline and the open/closed differential cannot be re-run; an occupancy characterization was done instead (differentiation reshapes accessibility more than genotype). The DHB-Venus ImageJ tool (C12) is public+present (MIT) with example data; its non-bundled plugin deps were resolved (BioVoxxel) and GUI/Windows-path bugs patched, but the interactive macro hangs in pure headless mode (no xvfb) -- and it maps to no deposited paper figure anyway. NOT attempted: LISA TF-enrichment (external Cistrome DB). All compute ran on «our HPC» SLURM compute nodes; data on «infra»; small results + scripts + logs saved under reproduction/.
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 50assessed: 2026-06-19 ⛓ 4420c2e74b5b
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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
- 2026-06-29
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no human curator yet
- 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 cyclin-dependent kinase inhibitor p27 (Cdkn1b), beyond its canonical cell-cycle role, non-canonically regulates gene expression programs (via SOX2 and its downstream targets OLIG2/ASCL1) to coordinate cell cycle exit with differentiation in adult subependymal neurogenesis and oligodendrogenesis.
- ★ p27 restricts residual CDK activity after mitogen withdrawal to antagonize cell cycling, but is not essential for cell cycle exit per se finding
- ★ p27 is required to repress genes involved in the transit from multipotentiality to differentiation, including the progenitor transcription factors Sox2, Olig2 and Ascl1 finding
- ★ p27 directly associates with regulatory sequences (promoters/enhancers) in the Sox2, Olig2 and Ascl1 genes mechanism
- ★ p27 repression of Sox2 hierarchically leads to reduced levels of its downstream targets Olig2 and Ascl1 mechanism
- ★ In vivo, p27 is required for setting proper SOX2 levels so neuroblasts and oligodendroglial progenitor cells timely exit the cell cycle, in a lineage-dependent manner finding
- ★ p27-deficient NPCs undergo extra cell divisions but eventually fully differentiate, with a bias toward more neurons and oligodendrocytes but not astrocytes finding
- Integrated ATAC-seq and RNA-seq profiling identifies p27-dependent chromatin accessibility and gene expression changes at the onset of NPC differentiation method
- ★ A CDK2 activity reporter (DHB-mVenus) shows p27-deficient cells retain higher residual CDK2 activity at mitogen withdrawal, reducible to WT levels by a CDK1/2 inhibitor finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| ATAC-seq | adult SEZ-derived NSC/NPC neurosphere cultures (WT and p27KO) | mitogen withdrawal (differentiation induction) | genome-wide promoter chromatin accessibility (Open/Closed) | — |
| bulk RNA-seq | adult SEZ-derived NSC/NPC neurosphere cultures (WT and p27KO) | mitogen withdrawal (differentiation induction) | differential gene expression (UP/DOWN) | — |
| LISA integrative TF motif/regulatory modeling (using public MNase-seq/ChIP-seq datasets) | computational analysis of RNA-seq/ATAC-seq gene lists | none | transcription factors associated with Closed/differentially regulated gene promoters | LISA bioinformatic tool |
| HOMER motif discovery | computational analysis of Closed+DOWN gene promoter sequences | none | enriched TF binding motifs | HOMER |
| Immunocytochemistry | NSC/NPC cultures (WT and p27KO) | mitogen withdrawal; p27 cDNA re-expression (p27-Flag) in KO | percentage/intensity of p27, Ki67, SOX2, OLIG2, ASCL1, βIII-tubulin, GFAP, O4 positive cells | — |
| EdU incorporation assay | NPC cultures (WT and p27KO) | mitogen withdrawal | percentage of EdU+ (S-phase) cells | — |
| Live-cell CDK2 activity reporter imaging (DHB-mVenus) | NPC cultures transduced with CSII-EF-DHB-mVenus reporter (WT and p27KO) | mitogen withdrawal ± CDK1/2 pharmacological inhibitor | cytoplasm/nucleus fluorescence ratio (CDK2 activity) | CSII-EF-DHB-mVenus reporter, bioimage analysis |
| Chromatin immunoprecipitation (ChIP) | WT NPC cultures | proliferation vs onset of differentiation | p27 occupancy at Sox2, Sox2 SRR2, Olig2 and Ascl1 regulatory regions (qPCR, IP/NRA ratio) | anti-p27 ChIP-qPCR |
- ▼ 85% reduction in the number of genes with differentiation-associated changes in chromatin accessibility or expression in p27KO vs WT cultures 85% reduction
- ▼ 88% of DE genes showing Closed chromatin during WT differentiation lost this change in p27KO cells 88%
- ▲ p27KO cultures show increased EdU incorporation at 2+1 DIV compared to WT, indicating extra cell cycling after mitogen withdrawal 9.1% ± 2.0 (n=5) vs 2.2% ± 0.4 (n=6)
- ▲ p27 binds Sox2 promoter/SRR2 enhancer and Olig2/Ascl1 promoters in proliferating cells, with binding strengthened at onset of differentiation IP/NRA ratio 1.82-3.70 (proliferation), 2-3-fold increase at differentiation
- ▼ SOX2+OLIG2+ASCL1+ triple-positive cell proportion decreases from proliferation to onset of differentiation in WT cultures 37.3% ± 1.7 to 19.5% ± 2.7 (n=9)
- ▲ p27KO cultures show significantly higher proportion of cells with elevated ASCL1, OLIG2 and SOX2 at onset of differentiation, restored to WT levels by p27 cDNA re-expression
- – p27KO cultures fully differentiate by 2+5 DIV but with a bias toward more neurons and oligodendrocytes, not astrocytes, relative to WT
- ▲ p27-deficient cells show higher residual cytosolic DHB-mVenus signal (CDK2 activity) at 2+1 DIV, normalized to WT levels by CDK1/2 inhibitor treatment
- pvalue p = 0.0015 (EdU+ cells p27KO (9.1±2.0%, n=5) vs WT (2.2±0.4%, n=6) at 2+1 DIV)
- fold_change 85% reduction (number of genes with expression/accessibility changes in p27KO vs WT during differentiation)
- fold_change 88% (proportion of Closed+DE gene changes in WT lost in p27KO)
- pvalue p = 0.008 (p27), p = 0.016 (Ki67) (repeated measures one-way ANOVA for percentage of p27+ and Ki67+ cells during WT differentiation)
- mean 37.3% ± 1.7 (2 DIV) vs 19.5% ± 2.7 (2+1 DIV) (percentage of SOX2+OLIG2+ASCL1+ triple-positive cells, p = 0.0015)
- other ChIP ratio p27-IP/NRA control: 1.82-3.70 (proliferation), strengthened 2- to 3-fold at differentiation (p27 binding to Sox2, Olig2, Ascl1 regulatory regions)
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 combines cell-culture-based quantitative assays (immunocytochemistry percentages/intensities, EdU incorporation, a CDK2-activity bioimage reporter) analyzed with ANOVA and pairwise comparisons, with genome-wide ATAC-seq/RNA-seq profiling of wild-type versus p27KO cells analyzed via FDR-controlled differential expression/accessibility calls and bioinformatic TF/motif enrichment tools (LISA, HOMER). Results from cell-based assays are reported as mean ± s.e.m. with exact p-values and significance asterisks, with biological replicates shown as individual dots on graphs.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Repeated measures one-way ANOVA | Fig. 1e, percentage of p27+ and Ki67+ cells across differentiation timepoints | — | not stated |
| Unspecified statistical test (comparison of two group means, p=0.0015) | EdU-incorporating cell proportions, p27KO vs wild-type at 2+1 DIV | n=5 (p27KO) vs n=6 (wild-type) | not stated |
| Unspecified statistical test (comparison of two group means, p=0.0015) | Fig. 3d, percentage of SOX2+OLIG2+ASCL1+ triple-positive cells, 2 DIV vs 2+1 DIV | n=9 | not stated |
| FDR-controlled differential expression/accessibility analysis (sFDR) | Genome-wide RNA-seq and ATAC-seq comparisons, Fig. 1b–c, 2f–h | — | not stated |
| LISA integrative TF-association analysis | Identification of TFs regulating Closed/DE gene sets, Fig. 1d, Fig. 3b | — | not stated |
| HOMER motif enrichment analysis (FDR<0.05) | TF binding motif enrichment in gene promoters, Fig. 1d, Fig. 3c | — | not stated |
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Repeated measures across differentiation timepoints (Fig. 1e) were analyzed with a repeated measures one-way ANOVA.↳ Could also: A linear mixed-effects model — Mixed-effects models can accommodate unbalanced replicate numbers or missing timepoints while still accounting for within-culture correlation across repeated measurements.
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Two-group comparisons (e.g., EdU incorporation, triple-positive cell percentages) appear to use an unspecified parametric test for small sample sizes (n=5–9).↳ Could also: A nonparametric test such as the Mann-Whitney U test — With small biological replicate numbers, distributional assumptions underlying parametric tests can be harder to verify, and a rank-based test provides an alternative that does not assume normality.
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Variability in quantitative measurements is summarized using standard error of the mean (s.e.m.).↳ Could also: Reporting standard deviation (SD) or a 95% confidence interval — SD directly conveys the spread of the underlying biological replicates, and a 95% CI additionally communicates the precision and plausible range of the estimated effect, which some readers find complementary to a p-value.
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Genome-wide differential expression and accessibility analyses use an FDR-controlled threshold, described as "sFDR" without naming the specific procedure.↳ Could also: Explicitly specifying and citing the FDR procedure (e.g., Benjamini-Hochberg) — Naming the exact multiple-testing correction procedure can aid reproducibility and let readers relate the reported FDR threshold to a specific, well-characterized statistical method.
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Multiple individual cell-based comparisons are performed across many figure panels within the same experimental series.↳ Could also: A family-wise error correction (e.g., Holm-Bonferroni) applied across the full set of related comparisons in a figure — When many hypothesis tests are shown together, a family-wise correction can help manage the cumulative probability of false-positive findings across the whole comparison set.
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Statistical significance is conveyed primarily via p-values and threshold asterisks.↳ Could also: Reporting standardized effect sizes (e.g., Cohen's d) alongside p-values — Effect sizes provide a measure of the magnitude of the difference independent of sample size, which can complement significance testing, particularly for small-n biological replicate studies.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-36627412
Paper: Domingo-Muelas et al. 2023, Cell Mol Life Sci 80:36. "The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2." PMID 36627412 · PMCID PMC9832098 · DOI 10.1007/s00018-022-04676-6.
Artifacts available
| Artifact | What it is | Where | Status |
|---|---|---|---|
paucabar/DHB-Venus |
ImageJ macro DHB-Venus.ijm + CellProfiler .cpproj + shipped example confocal dataset (Olympus .oif). CDK2-activity (nucleus/cytoplasm ratio) image-analysis tool. |
GitHub, MIT, commit 542371fb79a14e7317cfe1b6860af638bda2cc81 (2019-09-30, master) |
public, runnable |
| GSE196328 | RNA-seq of WT & p27KO, 10 samples (GSM5870569–578). SuperSeries member. Supplementary = gene-level raw read count matrix GSE196328_RNAseq_p27_raw_reads_GEO.txt.gz. |
GEO, public | obtainable |
| GSE196285 | ATAC-seq of WT & p27KO, 10 samples (GSM5866907–916). Supplementary = GSE196285_RAW.tar. |
GEO, public | obtainable |
| GSE196329 | SuperSeries wrapping the two above. | GEO, public | obtainable |
MOESM1_ESM.xlsx |
Supplementary Tables S1+S2 = LISA TF-enrichment p-values on Closed gene sets. | Springer ESM (saved in original/) |
downloaded |
Methods (verbatim-derived, pipeline tools)
- RNA-seq: raw reads → STAR align to Ensembl mouse transcriptome → RSEM count raw reads/gene → filter genes <1 CPM in ≥4 samples → coverage via deepTools bamCoverage (RPKM) → DESeq2 DE, FDR 0.05 up/down. ngsplot metagene/TSS heatmaps.
- ATAC-seq: FastQC/MultiQC → Bowtie2 map to GRCm38/mm10 (mate dovetailing) → Picard MarkDuplicates → SAMtools CollectInsertSizeMetrics → MACS2 peak calling (narrow, no model, FDR 0.05) → deepTools coverage → DiffBind differential peaks (FDR 0.05; open/closed) → ChIPseeker annotation (Gencode M18, ±3 kb TSS, 5 kb flank).
- Integration: DiffBind + DESeq2 tables combined in R; genes labeled by Closed_UP/Closed_DOWN/Open_UP/Open_DOWN (Fig 1b/c bubbles + scatter).
- TF enrichment: LISA (Cistrome) on Closed gene lists → Tables S1/S2.
- CDK2 imaging: custom ImageJ workflow = the
DHB-Venusrepo (nucleus & cytoplasm binary masks per cell from DAPI + DHB-mVenus channels → nucleus/cytoplasm intensity ratio).
IN SCOPE (pipeline-derived, reproduction targets — priority order)
- RNA-seq DESeq2 differential expression from the provided gene-level count matrix (GSE196328). Skips STAR/RSEM (authors shipped the counts). Reproduce: CPM filter (≥1 CPM in ≥4 samples), DESeq2 DE for the differentiation comparison (PRO vs 2+1 DIV) and the genotype comparison (WT vs p27KO); DEG counts at FDR<0.05; direction/significance of key genes Sox2, Olig2, Ascl1, Cdkn1b(p27). Headline molecular claim: p27 represses Sox2→Olig2/Ascl1 (de-repressed in p27KO).
- DHB-Venus ImageJ macro on the repo's shipped
example_dataset— run the authors' own published code headless (Fiji) and reproduce the nucleus/cytoplasm ratio results table (tool-level reproducibility; the paper's own images are wet-lab and not deposited, so this validates the code, not the paper figure). - ATAC-seq Bowtie2→MACS2→DiffBind from GSE196285 RAW (stretch; heavier: genome index + alignment). Differential (open/closed) peak counts at FDR 0.05.
OUT OF SCOPE (wet-lab / manual / external — not attempted)
- All immunostaining/IHC cell counts, flow cytometry / FACS sorting, p27-ChIP-qPCR, Western blots, qRT-PCR, in-vivo OB/SEZ neurogenesis quantifications, EdU/Ki67 scoring (manual counting "using Olympus and ImageJ/Fiji").
- LISA TF-enrichment exact p-values (Tables S1/S2): depends on Cistrome DB + the authors' exact upstream gene lists; recorded as reported claims but reproduction is low-tractability (external DB), attempted only if primary targets succeed.
- "deepTools/ngsplot/IGV" visualization tracks (qualitative, no numeric claim).
Assessments & scoring basis
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.