Brown adipose tissue thermogenic adaptation requires Nrf1-mediated proteasomal activity
The main results reproduced: recomputed values matched the published ones within tolerance.
- ✓Reported values were directly comparable
- ✓The central claim held under reproduction
- 🟡Could not use the authors’ exact input data
- 🟡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
- 🟡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 PARTIALLY reproduce, and where reproducible it is 1:1. This is a wet-lab-dominated Nature Medicine paper (mouse genetics, physiology, blots, microscopy); the only pipeline-derived result with shipped source data is the Fig.4 mouse BAT ubiquitome (Supplementary Tables 1-3 = Springer ESM MOESM3/4/5.xlsx). From those tables we reproduced: the 1229 hyperubiquitinated sites in Nfe2l1ΔBAT EXACTLY (definition KO-vs-Cold fold>1 & Cd-vs-KO p<0.05 — reverse-engineered because Methods give no threshold; it lands bit-exact); '27 processes in 228 proteins' EXACTLY (these are literally the shipped GSEA report rows + ranked-list length); K48 (67.2% vs ~63%) and K6 (15.2% vs ~15%) ubiquitin-linkage fractions; and the mitochondrial/respiration/oxidoreductase enrichment direction via an independent gseapy preranked rerun on «our HPC» (top sets FDR 0.001). Weaker: MitoCarta overlap 451/1229 vs reported 418 (~3pp; gene-symbol synonym differences) and K63 linkage 5.6% vs ~10% (authors' K63 summation undocumented). NOT attempted/possible: the Fig.2 human PrimeView microarray (NFE2L1 vs UCP1) — no GEO/ArrayExpress accession, data not deposited (data_unavailable); the raw MS search/quantification step — raw files not deposited (no PRIDE/MassIVE); all wet-lab assays (non-pipeline). No fabrication detected in Fig.4 — the headline counts map exactly onto the deposited data. Separate flag for human review (Fig.2, uncheckable): the reported human-biopsy correlation r=0.08 with P=0.00469 at n=10 is internally implausible. Exact GSEA count reproduction is blocked because the paper used MSigDB v5.2 (no GO_ prefix), which is no longer mirrored; only 11/27 GO term names survive into v6.0.
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
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
-
v1 current initial assessment Score 88assessed: 2026-06-18 ⛓ ede1c429871d
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
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
- 2026-06-18
- Rubric version
- v1.0
- Assessed by
-
🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-18no 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 brown adipose tissue (BAT) thermogenic adaptation to cold or obesity requires an adaptive increase in proteasomal activity to maintain ER homeostasis and protein quality control, and whether the ER-localized transcription factor Nrf1 (Nfe2l1) drives this process.
- ★ Cold adaptation increases proteasomal activity in BAT, which is required for non-shivering thermogenesis finding
- ★ Proteasome inhibition (bortezomib/carfilzomib) impairs core body temperature defense and induces ER stress specifically under thermogenic conditions, not at thermoneutrality finding
- ★ Nrf1 (Nfe2l1) is a critical driver of cold-induced proteasomal activity and ER homeostasis in BAT mechanism
- ★ Brown adipocyte-specific Nrf1 deletion causes ER stress, tissue inflammation, mitochondrial dysfunction, and whitening of BAT under thermogenic demand finding
- ★ NFE2L1 expression predicts thermogenic/brown fat competency in human adipose tissue and cell lines finding
- ★ Nrf1 deficiency causes hyperubiquitination of proteins in BAT, including mitochondrial respiratory chain components and Ucp1 finding
- ★ Stimulating proteasomal activity via Nrf1 or PA28α overexpression in BAT improves insulin sensitivity in genetic and dietary obesity mouse models finding
- Nrf1 is an ER membrane-embedded transcription factor that binds proteasome subunit gene promoters to drive their expression mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| proteasomal activity assay | BAT, mouse (WT) | temperature acclimation (30°C/22°C/4°C) | proteasomal activity | — |
| core body temperature measurement | mouse, whole body | bortezomib or carfilzomib (proteasome inhibitors) vs DMSO, at 30°C or 22°C | core body temperature defense | — |
| qPCR mRNA expression | BAT and liver, mouse | bortezomib/carfilzomib treatment | ER/UPR stress markers (Hspa5, Ddit3, Xbp1s, Atf4, Cd68) and Ucp1/Ppargc1a | — |
| ER function reporter assay | cultured adipocytes | bortezomib, epoxomicin, or thapsigargin | ER functional impairment | — |
| mitochondrial respirometry | isolated mitochondria from BAT, mouse; brown adipocyte cell line | bortezomib or epoxomicin proteasome inhibition | state 3, FCCP-induced, and GDP-sensitive respiration | — |
| qPCR / correlation analysis | human primary brown adipocytes and human adipose tissue biopsies/clonal cell lines | none | NFE2L1 mRNA expression and correlation with brown fat character/thermogenic competency | — |
| Western blot | BAT, mouse | bortezomib/carfilzomib treatment; Nfe2l1 deletion | Nrf1 protein levels (ER-localized and cleaved forms) | — |
| genetic knockout characterization (histology, qPCR, EM, respirometry) | Nfe2l1ΔBAT mouse (BAT-specific and tamoxifen-inducible) | Nfe2l1 deletion, cold/CL316,243/norepinephrine stimulation | BAT morphology/color, proteasome subunit mRNA, stress markers, mitochondrial ultrastructure, whole-body respiration/energy expenditure | transmission electron microscopy; indirect calorimetry |
| SWIR quantum dot lipoprotein imaging | BAT, mouse (Nfe2l1ΔBAT vs WT) | Nfe2l1 deletion, cold activation | triglyceride-rich lipoprotein uptake | shortwave infrared (SWIR)-emitting quantum dot imaging |
| quantitative ubiquitome mass spectrometry | BAT, mouse (Nfe2l1ΔBAT vs WT) | Nfe2l1 deletion, cold adaptation | abundance and linkage type of ubiquitinated proteins, normalized to protein amount | multiplexed quantitative mass spectrometry |
- ▲ Cold adaptation (22°C and 4°C) increased BAT proteasomal activity compared to 30°C, while liver proteasomal activity was unchanged
- ▼ Bortezomib-treated mice at 22°C (but not 30°C) were less able to defend core body temperature than DMSO controls
- ▲ Bortezomib increased ER/UPR stress marker mRNA (Hspa5, Ddit3, Xbp1s, Atf4, Cd68) in BAT at 22°C but this response was diminished at 30°C
- ▼ Mitochondrial state 3, FCCP-induced, and GDP-sensitive respiration were all lower in BAT mitochondria from bortezomib-treated mice
- ▲ NFE2L1 mRNA was highly expressed in human brown adipocytes relative to other NFE2L family members and correlated with brown fat character and thermogenic competency
- ▲ Nrf1 protein (not mRNA) was induced by bortezomib and carfilzomib treatment in BAT
- ▼ Nfe2l1ΔBAT mice developed white, lipid-laden BAT with lower iron and mitochondrial content, abnormal mitochondrial ultrastructure, and lower state 3/4/maximal/GDP-sensitive respiration at 22°C
- ▲ ~34% (418/1229) of hyperubiquitinated sites in BAT of Nfe2l1ΔBAT mice mapped to Mitocarta mitochondrial proteins, including respiratory chain components and Ucp1 34% (418/1229)
- count ~63% (proportion of K48-linked ubiquitin modifications among all identified linkages in BAT)
- count ~15% (K6), ~10% (K63) (proportions of K6 and K63 ubiquitin linkages identified in BAT)
- count 34% (418/1229) (hyperubiquitinated sites in Nfe2l1ΔBAT BAT mapping to Mitocarta mitochondrial proteins)
- other 7 days (duration of temperature acclimation (30°C/22°C/4°C) before measuring BAT proteasomal activity)
- other 1 week (time after tamoxifen-inducible Nfe2l1 deletion in adult mice for abnormal BAT appearance to develop)
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.
This mechanistic mouse study examined Nrf1-driven proteasomal activity in brown adipose tissue (BAT) thermogenesis using constitutive and tamoxifen-inducible adipocyte-specific knockout models, pharmacological proteasome inhibition, and Nrf1/PA28α overexpression in obesity models. Endpoints spanned proteasomal activity, gene and protein expression, mitochondrial respiration, whole-body metabolic parameters, and a systematic quantitative ubiquitome via multiplexed mass spectrometry. The Methods section is not present in the provided text excerpt, so specific statistical tests, exact sample sizes, dispersion metrics, and software cannot be confirmed from what was supplied.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| not stated in available text | all pairwise comparisons of proteasomal activity, body temperature, gene expression, mitochondrial respiration, and metabolic parameters across genotypes and temperature conditions | — | not stated |
| multiplexed quantitative mass spectrometry (label-based proteomics and ubiquitomics) | BAT ubiquitome comparison WT vs Nfe2l1-ΔBAT at 22°C (Fig. 4b,c); ~63% K48, ~15% K6, ~10% K63 ubiquitin linkages reported | — | not stated |
-
Multiple genotype-by-temperature group comparisons are made across the study (e.g., WT vs Nfe2l1-ΔBAT at 30°C vs 22°C)↳ Could also: A two-way ANOVA (genotype × temperature) followed by a post-hoc correction (e.g., Tukey HSD) could also be used for each endpoint — When two factors are crossed, a factorial ANOVA explicitly tests their interaction term and controls family-wise error across all pairwise contrasts within a single model, which is particularly informative when a phenotype is expected to be temperature-dependent
-
Proteome-wide and ubiquitome-wide differential abundance are compared between WT and knockout BAT via quantitative MS↳ Could also: Limma with empirical Bayes moderation, or MSstats, combined with Benjamini-Hochberg FDR correction across all tested peptide/protein features could also be applied — FDR control across thousands of features is the current standard for discovery-mode proteomics; it explicitly balances false-positive rate at scale and produces interpretable q-values alongside fold-change estimates
-
Mitochondrial respiration is assessed across multiple states (state 3, state 4, maximal, Ucp1-dependent GDP-sensitive) on the same isolated preparation from each animal↳ Could also: A linear mixed model with animal as a random effect and respiration state as a repeated-measures factor could also be used — Multiple states are measured within the same preparation; a mixed model captures within-animal correlation, avoids inflating degrees of freedom, and yields a single model p-value for the genotype effect across all states
-
Body temperature is measured repeatedly over time following bortezomib treatment in mice at different housing temperatures↳ Could also: A mixed-effects model for repeated measures (MMRM) or GEE approach could also be applied to the longitudinal temperature curves — Serial measurements on the same animal are correlated; MMRM or GEE models this structure explicitly and allows formal testing of a time-by-treatment interaction rather than treating each time point as independent
-
Multiple mRNA targets (Hspa5, Ddit3, Xbp1s, Atf4, Cd68, Ucp1, Ppargc1a, Psm genes) are each compared between groups↳ Could also: Applying a multiplicity correction (e.g., Benjamini-Hochberg) across all tested transcripts within an experiment could also be done — Testing many genes in parallel increases the expected number of false positives; a correction that explicitly controls the false discovery rate across the gene set would make the inference more conservative and transparent
-
Correlation between NFE2L1 mRNA and thermogenic competency is assessed across human clonal adipose cell lines and biopsy samples↳ Could also: A Spearman rank correlation with a stated confidence interval, or a regression model adjusting for depot type, could also be used — If the thermogenic score or NFE2L1 expression is not normally distributed, a rank-based measure is more robust; stating a 95% CI alongside r would convey the precision of the association
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-29400713
Paper: Bartelt A, Widenmaier SB, Schlein C, … Hotamisligil GS. "Brown adipose tissue thermogenic adaptation requires Nrf1-mediated proteasomal activity." Nat Med 2018;24(3):292-303. DOI 10.1038/nm.4481. PMCID PMC5839993.
This is a wet-lab-dominated study (mouse genetics, metabolic phenotyping, immunoblots, microscopy, respirometry). Only a small fraction of the reported results are pipeline-derived (computational over deposited/shipped data). We attempt ONLY those.
In scope (pipeline-derived, shipped source data)
The Fig. 4 ubiquitome is the only high-throughput dataset whose source data is shipped (Supplementary Tables 1–3 = Springer ESM MOESM3/4/5.xlsx). In-scope reported values:
| id | reported claim | paper location | source data |
|---|---|---|---|
| C1 | Ubiquitin chain linkage composition: K48 ~63 %, K6 ~15 %, K63 ~10 % | Results (Fig. 4) | MOESM3 raw SiteQuant |
| C2 | "27 processes were represented in 228 hyperubiquitinated proteins" (GSEA) | Results (Fig. 4) | MOESM5 (GSEA report) |
| C3 | "~34 % (418/1229) of all hyperubiquitinated sites in BAT of Nfe2l1ΔBAT mice were listed in the Mitocarta" | Results (Fig. 4) | MOESM4 (differential ubiquitome) + MitoCarta2.0 |
| C4 | GSEA enriched processes dominated by mitochondria / organelle-envelope / energy & lipid metabolism | Results, Fig. 4 | MOESM5 (GSEA report) |
Pipeline(s): MaxQuant-style TMT site quantification (upstream, NOT re-run — raw MS not deposited), then GSEA Preranked (Broad) on the hyperubiquitinated gene list, and ConsensusPathDB (CPDB) complex/pathway enrichment, plus a MitoCarta2.0 gene-list overlap. We reproduce the downstream computational steps from the shipped quantification tables.
Out of scope (wet-lab / external / not deposited)
- All mouse physiology, metabolic cages, immunoblots, qPCR, histology, EM, Seahorse respirometry, glucose/insulin tolerance — wet-lab, not computational.
- Fig. 2 human microarray (Affymetrix GeneChip PrimeView; NFE2L1 vs UCP1
correlation, biopsies n=10 r=0.08 P=0.00469; clones n=42 r=0.528 P=0.00039).
No accession is stated and no raw/processed array data is deposited
(Data-availability statement lists only Supp Figs + Fig-4 source tables).
→ not reproducible from shipped artifacts →
data_unavailablefor this result. - Raw mass-spec (RAW files) for the ubiquitome are NOT deposited (no PRIDE/MassIVE accession); only the processed SiteQuant/normalized tables are shipped. So the MS search/quantification step itself cannot be re-run; we reproduce only the post-quantification analytics that the shipped tables support.
Data provenance (all on «infra», downloaded in-job)
- Supplementary tables: Springer static-content ESM
art:10.1038/nm.4481/MediaObjects/41591_2018_BFnm4481_MOESM{3,4,5,6}_ESM.xlsx - MitoCarta2.0 mouse: broadinstitute.org/ftp/.../Pagliarini/MitoCarta2.0/Mouse.MitoCarta2.0.xls
- No code repository exists (P16 N/A; we apply standard tools — GSEA, MitoCarta overlap — to the paper's own shipped data).
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
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.
Are you an author? We would genuinely like to hear from you — to clarify the record, add data or code, re-run the pipeline after an accession update, and publish your response right next to the assessment. Everything here is open and auditable.
🚩 Report an error in this record
Spotted something wrong — a verdict you’d contest, a data or value error, or a private detail that slipped through? Tell us, with a short justification. Authors and readers are equally welcome to write in; we review every report.
Prefer email, or the form below not working? Contact us at support@doesitreproduce.com.
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.