TFEB deficiency attenuates mitochondrial degradation upon brown adipose tissue whitening at thermoneutrality
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”.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- 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
▸Reproduction agent’s raw note
DROP (non_pipeline). 'TFEB deficiency attenuates mitochondrial degradation upon brown adipose tissue whitening at thermoneutrality' (Sass, Schlein et al., Molecular Metabolism 2021;47:101173, DOI 10.1016/j.molmet.2021.101173, PMC7903014) is a wet-lab mouse-physiology study. It is NOT described well enough to reproduce computationally because there is nothing computational to reproduce: gene expression is targeted qPCR (no RNA-seq/microarray); the mitochondria-lysosome co-purification is read out by subcellular fractionation + Western blot (no proteomics/mass-spec); all other data are histology/EM/immunofluorescence, indirect calorimetry, OGTT, body composition and metabolic-tracer assays. There is no data-availability statement and no public accession (GEO DataSets query for PMID 33516944 = 'No items found'; no GEO/SRA/ArrayExpress/PRIDE/figshare/zenodo), and no analysis-code repository (own or third-party). The only software named (ImageJ/Adiposoft, Image Studio Lite densitometry, GraphPad Prism 8, Excel, BioRender) are off-the-shelf GUI tools acting on raw images/Ct values that were never deposited, so even the lone image-analysis step (Adiposoft lipid-droplet sizing) is uncheckable. The brief's P16 'apply a third-party tool to the paper's own data' path is blocked because the paper deposited no data of any kind. Genuinely verified via full text (PMC7903014), GEO, and web search; no compute submitted to «our HPC» because there is nothing to run. NOT ATTEMPTED: any 1:1 numeric reproduction (impossible without raw data/code). Honest drop per BRIEF rule 6; no value fabricated. Note: operator Christian Schlein is a co-author.
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.
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v1 current initial assessmentassessed: 2026-06-18 ⛓ 130d10eee2f3
✎ 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
- 2026-07-15
- Rubric version
- v1.0
- Assessed by
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🤖 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: sonnetTFEB, as the only MiT-family transcription factor upregulated during brown adipose tissue (BAT) whitening, drives whitening-associated mitochondrial breakdown at thermoneutrality via the autophagosomal/lysosomal machinery.
- ★ BAT whitening at thermoneutrality is associated with TFEB-dependent mitochondrial degradation. finding
- ★ Brown adipocyte-specific TFEB-KO BAT retains higher mitochondrial mass/protein levels (UCP1, respiratory chain complexes) upon thermoneutral adaptation, independent of increased mitochondriogenesis. finding
- ★ Preserved mitochondrial mass in TFEB-KO mice does not translate into elevated thermogenic capacity or protection from diet-induced obesity. finding
- ★ TFEB is dispensable for thermogenic adipose tissue function under cold exposure. finding
- ★ Mitochondria in thermoneutral TFEB-KO BAT are presumably trapped in late stages of mitophagy (lysosomes/autophagosomes), as indicated by co-localization/co-purification of lysosomal markers with mitochondria. mechanism
- Generation of a brown adipocyte-specific TFEB knockout mouse model (TFEBfl/fl x Ucp1-Cre). resource
- ★ Homeostatic expression of lysosomal (CLEAR network) genes in BAT is likely mediated by transcription factors other than TFEB, since known TFEB target gene expression was only modestly affected by TFEB deficiency. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| qRT-PCR gene expression analysis | BAT/WAT tissue and primary brown adipocytes, TFEB-BAT-KO vs WT mice | KO (brown adipocyte-specific TFEB deletion) | mRNA levels of lysosomal/autophagy, mitochondrial, and thermogenic genes | 7900HT Sequence Detection System (Applied Biosystems) |
| Western blot / immunoblotting | BAT tissue and primary brown adipocytes | KO; chloroquine treatment (autophagic flux) | Protein levels of UCP1, OXPHOS complexes, LC3B, p62, LAMP1, Cathepsin D, PINK1, Parkin, VDAC, HSL/p-HSL | — |
| Immunohistochemistry/confocal microscopy | BAT tissue sections | KO | Co-localization of LC3B/lysosomal puncta with mitochondria (blinded puncta counting) | Nikon eclipse Ti confocal laser scanning microscope |
| Histology (H&E staining) | Subscapular BAT tissue | KO; thermoneutral housing | Tissue morphology and lipid droplet size | Nikon eclipse Ti microscope with DS-Fi2-U3 camera; Adiposoft (ImageJ) |
| Indirect calorimetry / body temperature telemetry | Whole mouse (TFEB-BAT-KO vs WT) | Cold exposure (6/22/30°C) and CL316,243 injection | Energy expenditure, respiratory quotient, body temperature | TSE PhenoMaster system; G2 E-Mitter telemetric transponders |
| Metabolic tracer study | Whole mouse, metabolically active tissues | Oral gavage of 3H-triolein and 14C-/3H-glucose | Tissue uptake of lipids and glucose | Tri-Carb scintillation counter |
| Oral glucose tolerance test and plasma biochemistry | HFD-fed TFEB-BAT-KO vs WT mice | High-fat diet feeding, KO | Blood glucose, plasma insulin, cholesterol, triglycerides, NEFA | AccuCheck Aviva; Chrystal Chem insulin kit; Roche/Wako colorimetric kits |
| Mitochondrial isolation and immunoblotting | Isolated BAT (iBAT/scBAT) mitochondria, pooled from 5 mice | Fasting/refeeding at 30°C, KO | Mitochondrial protein content/localization | — |
- ▲ TFEB-KO BAT preserves mitochondrial mass and UCP1/respiratory chain complex protein levels under thermoneutral conditions, independent of mitochondriogenesis.
- – Higher mitochondrial/UCP1 levels in TFEB-KO mice did not translate into elevated thermogenic capacity or protection from diet-induced obesity.
- ▼ TFEB-KO mice showed a lower metabolic (energy expenditure) response to CL316,243 injection compared to WT.
- – Autophagosomal/lysosomal marker levels are altered in TFEB-deficient BAT and primary adipocytes.
- ▲ Lysosomal markers co-localize and co-purify with mitochondria in TFEB-deficient BAT.
- – Known TFEB target gene expression is only modestly affected by TFEB deficiency in BAT.
- – TFEB is dispensable for thermogenic adipose tissue function during cold exposure.
- other 60% calories from fat, 16 weeks (High-fat diet protocol for diet-induced obesity study)
- other 1 mg/kg body weight (CL316,243 dose used to induce non-shivering thermogenesis)
- other 50 μM, 2 h (Chloroquine treatment dose/duration for in vitro autophagic flux assay)
- other 3H-triolein 0.72 MBq/kg; 14C-glucose 0.15 MBq/kg (Tracer doses for lipid and glucose uptake study)
- count 5 mice pooled (Mice pooled per sample for BAT mitochondria isolation)
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 study used a two-group (TFEB BAT KO vs. TFEB BAT WT littermate controls) mouse design with brown adipocyte-specific Cre-lox deletion, assessing BAT whitening across housing temperatures (6°C, 22°C, 30°C) and a 16-week HFD protocol. Endpoints included qPCR, Western blot, indirect calorimetry, histomorphometry, immunofluorescence, and metabolic tracer studies. The statistical analysis subsection is not present in the provided text excerpt, so specific tests, software, and reporting conventions cannot be confirmed from the supplied material. Blinded counting by three independent observers was explicitly noted for LC3B puncta quantification.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| not stated in provided excerpt | all pairwise KO vs. WT comparisons across temperature and diet conditions | — | not stated |
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Two genotypes were compared across multiple housing temperatures (6°C, 22°C, 30°C) and a dietary condition (chow vs. HFD), constituting a multi-factorial design↳ Could also: Two-way ANOVA (genotype × temperature, or genotype × diet) with an appropriate post-hoc test (e.g., Tukey HSD or Sidak) could be used to jointly model both factors and their interaction in a single model — A factorial model explicitly tests whether the genotype effect on mitochondrial markers differs across thermal conditions (interaction term) and controls the family-wise error rate across all pairwise comparisons within one framework, rather than running separate tests per condition
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Body weight and metabolic parameters were tracked repeatedly over a 16-week HFD protocol within the same animals↳ Could also: Linear mixed-effects models or repeated-measures ANOVA explicitly accounting for the within-animal correlation structure over time could be used — Mixed models handle the non-independence of repeated measures on the same animal, can accommodate missing time points under a missing-at-random assumption, and allow random slopes per animal — all of which improve validity compared to treating each time point independently
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Indirect calorimetry-derived energy expenditure was compared between genotypes↳ Could also: ANCOVA with lean body mass (or total body mass) as a covariate is a commonly recommended approach in metabolic phenotyping literature — Raw energy expenditure scales with body size; if genotypes differ in body composition, an ANCOVA adjusts for the mass-dependent component and isolates the genotype effect on metabolic rate per se, which is the quantity of biological interest
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LC3B puncta were counted by three independent blinded observers and presumably averaged↳ Could also: Intraclass correlation coefficient (ICC) or Krippendorff's alpha could be computed across the three raters before aggregating counts — Reporting inter-rater reliability quantifies how much variance is attributable to observer disagreement versus true biological signal, strengthening confidence in the morphometric endpoint and aiding reproducibility by others
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Gene expression was normalized using the ΔΔCT method with a single housekeeping gene (Tbp or TfIIb)↳ Could also: Normalization to the geometric mean of two or more validated reference genes (per MIQE guidelines) is an alternative — A single reference gene may be co-regulated under the experimental conditions (thermoneutrality, TFEB deletion, differentiation state); using multiple stable reference genes reduces normalization error and is less susceptible to condition-dependent expression changes in any single gene
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For mitochondrial isolation, iBAT and scBAT from 5 mice were pooled into a single sample per condition↳ Could also: Preparing separate mitochondrial isolations from individual animals (or smaller pools) and treating each as an independent biological replicate would be an alternative — Pooling eliminates between-animal variance from the analysis; individual isolations would allow estimation of biological variability and enable formal statistical inference with animal-level replication, though this must be weighed against the practical yield constraints of the isolation protocol
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
scope.md — pmid-33516944
Title: TFEB deficiency attenuates mitochondrial degradation upon brown adipose tissue whitening at thermoneutrality Journal: Molecular Metabolism 2021;47:101173 · DOI 10.1016/j.molmet.2021.101173 · PMCID PMC7903014 · PMID 33516944 Authors: Sass F, Schlein C, Jaeckstein MY, Pertzborn P, Schweizer M, Schinke T, Ballabio A, Scheja L, Heeren J, Fischer AW (Heeren lab, UKE Hamburg) Note: Christian Schlein (the operator) is a co-author of this paper.
Verdict: DROP — non_pipeline
This publication is a wet-lab mouse physiology study. It contains no pipeline-derived computational result within the brief's scope, no deposited high-throughput dataset, and no analysis code repository. There is nothing to run on «our HPC» and nothing to reproduce 1:1.
Evidence (genuinely checked, not assumed)
Read in full: PubMed record, Europe PMC, PMC7903014 full text (Methods + figures + end matter), GEO DataSets, and a targeted web search.
| Check | Result |
|---|---|
| High-throughput sequencing (RNA-seq/scRNA/ChIP/ATAC) | None. Gene expression is targeted qPCR only ("7900HT Sequence Detection System … assays-on-demand"). |
| Microarray | None. |
| Proteomics / mass spec (incl. the mito–lyso co-purification) | None. Co-purification was analysed by subcellular fractionation + Western blot ("mitochondria … resuspended in RIPA … immunoblotting"), not LC-MS. No "mass spectrometry/proteomics/PRIDE" anywhere in the text. |
| Data availability statement / public accession | None. No GEO/SRA/ArrayExpress/PRIDE/figshare/zenodo accession in the paper. GEO DataSets query for PMID 33516944 → "No items found". |
| Analysis code repository (own or third-party) | None. No GitHub/GitLab/Zenodo code link. |
| Bioinformatic pipeline | None named. |
| Software actually used | ImageJ + Adiposoft plugin (lipid-droplet sizing), Image Studio Lite (blot densitometry), GraphPad Prism 8 (t-test / 1-way / 2-way ANOVA + Tukey), Microsoft Excel, BioRender (illustration). |
| Supplementary data | A single mmc1.docx (14.3 MB) of supplementary figures — not reusable raw data. |
In-scope vs out-of-scope (by result type)
ALL reported results are out of scope for pipeline reproduction:
- Body weight / composition, indirect calorimetry (energy expenditure), OGTT, plasma chemistry, ²-deoxyglucose / lipid tracer uptake → wet-lab in-vivo physiology (metabolic cages, injections). Out of scope; raw data not deposited.
- qPCR gene-expression panels → wet-lab; ΔΔCt computed in Excel/Prism on Ct values that are not deposited. Out of scope.
- Western blots + densitometry (incl. mito–lyso co-purification) → wet-lab; Image Studio densitometry on raw blot images that are not deposited. Out of scope.
- Histology (H&E), lipid-droplet size (Adiposoft), immunofluorescence, electron microscopy + mitochondrial morphometry → image analysis on raw microscopy images that were never deposited. Even the one image-analysis tool (Adiposoft) cannot be re-run because its inputs are absent. Out of scope / uncheckable.
Why not "apply a third-party tool to the paper's data" (P16)
The brief permits reproducing by running an existing third-party tool on the paper's own data. That path is blocked here because the paper deposited no data at all — no sequencing data, no proteomics, no raw images, no value tables. There is no input on which any tool could be run. This is therefore not a down-ranked own-vs-third-party case; it is a genuine absence of any reproducible computational artifact.
Drop record
drop_reason: non_pipeline (controlled vocab) — text-mining false positive; the title's "TFEB / mitochondrial degradation / whitening" reads genomics-like but the study is wet-lab physiology.- Supporting absences (any one alone would also block):
no_data_accession(no public dataset) andno_code(no analysis code). - No compute submitted to Hum
No individual results have been recorded for this entry yet.
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.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
This is a correct non_pipeline DROP. The paper (Sass, Schlein et al., Mol Metab 2021;47:101173) is a wet-lab mouse-physiology study with no deposited data (GEO query → 'No items found', no SRA/PRIDE), no analysis code, and only GUI tools (ImageJ/Adiposoft, Image Studio, Prism) operating on raw images/Ct values that were never shared. The blocker sits entirely on the data-availability/input side, not on a computational defect or fabrication — so q5 is red because values are not derivable from anything shared, but this is an availability issue, not an authors' integrity problem. The core claims cannot be put against any reproduced output, hence overall red for reproduction quality with no fabrication concern.
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
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.