Fig 2: Ubiquitinated Nur77 interacts with p62 to sequester damaged mitochondria.a Immunofluorescence images of GFP-Nur77, mCherry-p62, and HA-Ub transfected in HeLa cells treated with or without celastrol. Data illustrate the colocalization of Nur77 with p62 and Ub in the presence of celastrol. Scale bar, 10 µm. b, c Interaction of indicated Nur77 or deubiquitinated mutant (K536R) and p62 was analyzed in HeLa cells treated with or without celastrol by co-immunoprecipitation (co-IP) assay. d, e Immunofluorescence images showing the effect of celastrol-induced Nur77 ubiquitination on mCherry-p62 droplet formation. Scale bar, 10 µm. f Representative images showing ubiquitination-dependent colocalization of Nur77 with p62 and mitochondria in HeLa cells treated with celastrol. Scale bar, 10 µm. Data represent at least three independent experiments. Source data are provided as Source Data file.

Fig 3: Nur77 and p62 are required for celastrol-induced mitophagy.a Representative image of celastrol-induced mitophagy in HeLa, Nur77-/-HeLa, and Nur77-/-HeLa cells transfected with Myc-Nur77 by EGFP-mCherry-COX8 assay as described in Methods. Scale bar, 10 µm. b Representative images of celastrol-induced mitophagy in MEFs and p62-/-MEFs by EGFP-mCherry-COX8 assay as described in Methods. Scale bar, 10 µm. c Colocalization of Nur77, LC3, and p62 with mitochondria within mitophagosome/autolysosome. Upper panel: Electron micrographs of HeLa cells stained with 15 nm immunogold-conjugated Nur77 antibody to detect Nur77 (red), and 10 nm immunogold-conjugated p62 antibody to detect p62 (green). Bottom panel: Electron micrographs of HeLa cells stained with 15 nm immunogold-conjugated LC3 antibody to detect LC3, and 10 nm immunogold-conjugated p62 antibody to detect p62. Cells were treated for 1 h with celastrol. The blue dotted line indicates mitophagosome/autolysosome. Mito mitochondrion, Scale bar, 200 nm. d Representative images showing Hsp60, a mitochondrial marker, in the liver tissue from wild-type and Nur77-/-mice in aging model. Young mice, 8 weeks old. Aged mice, 2 years old. Scale bar, 10 µm. e Statistical analysis of mitochondrial size was represented from liver tissue. Left graph, n = 316, 253, 267, and 287, respectively; Right graph, n = 3 biologically independent samples. A two-tailed unpaired Student’s t-test was used for statistical analysis, and data were presented as mean values ± SEM. f The expression of Nur77 protein in the liver tissue from wild-type and Nur77-/-mice in the aging model. g Representative images of EGFP-mCherry-COX8 in the liver from wild-type or Nur77-/-mice in the aging model. Purple arrows indicate mitophagy. Scale bar, 2 µm. h Quantification of cells showing mCherry-COX8 accumulation on liver tissue. Two-tailed unpaired Student’s t-test was used for statistical analysis, and data were presented as mean values ± SEM (n = 5 mice per group). Data represent at least three independent experiments. Source data are provided as a Source Data file.

Fig 4: Nur77-LBD is insufficient to mediate celastrol-induced mitophagy.a–d Representative images showing colocalization of Nur77 or mutants with p62, mitochondria, and lysosome in HeLa cells after celastrol treatment. The blue arrow indicates line profiles of fluorescence intensities including Pearson’s correlation coefficients shown in b and d. A two-tailed unpaired Student’s t-test was used for statistical analysis, and data were presented as mean values ± SEM (n = 20 biologically independent samples). Dotted box: higher magnification of indicated region. Scale bar, 10 µm. e Mutating K536 in Nur77 inhibits celastrol-induced interaction between p62 and LC3. HeLa cells transfected with the indicated expression plasmids were treated with or without 2 µM celastrol and 20 ng/mL TNFa. Interaction of Flag-p62 with GFP-LC3 was examined by co-IP assay. f Characterization of domain requirement of Nur77 for promoting celastrol-induced p62 interaction with LC3. HeLa cells transfected with the indicated Flag-p62, GFP-LC3, and GFP-Nur77 or mutant were treated with celastrol and TNFa for 1 h and analyzed for Flag-p62 interaction with GFP-LC3 by co-IP assay. Data represent at least three independent experiments. Source data are provided as Source Data file.

Fig 5: Celastrol promotes phase separation and liquidity of p62.a Representative images showing the time-dependent effect on celastrol induction of cytoplasmic Nur77 body formation. Bottom panels: quantitative analysis of the number and size of Nur77/p62 body formation. Bottom left graph, n = 4 biologically independent samples; Bottom right graph, n = 20, 23, 25, and 19, respectively. Data were presented as mean values ± SEM. Scale bar, 10 µm. b Real-time images showing the formation and fusion of GFP-Nur77 and mCherry-p62 droplets in HeLa cells after treatment with celastrol (2 µM) for 1 h. White arrows indicate droplets formation and fusion (see also Supplementary Movie 1). Scale bar, 10 µm. c Representative images illustrating the role of celastrol in promoting p62 body formation in a Nur77-dependent manner immunostaining. Nur77-/-HeLa cells were also transfected with GFP-Nur77 to determine its effect on p62 body formation. The diameter of the biggest p62 puncta in each cell was measured. The number of p62 puncta >0.5 µm in each cell was assessed. A two-tailed unpaired Student’s t-test was used for statistical analysis, and data are presented as mean values ± SEM (n = 5 biologically independent samples). d FRAP analysis of the effect of Nur77 in regulating p62 mobility in HeLa cells. Data were presented as means ± SEM (n = 3 independent experiments). Scale bar, 1.5 µm. Data represent at least three independent experiments. Source data are provided as a Source Data file.