Fig 2: Role of NBR1 in adaptive thermogenesis in primary brown adipocytes.a qPCR analysis of Ucp1 expression in primary brown adipocytes differentiated from SVF of four indicated genotypes (n = 5 biological replicates). b Immunoblot analysis of UCP1 in primary brown adipocytes of four indicated genotypes, treated with ISO (0.5 µM) for indicated time. Representative blots (left) and quantification (right) from three independent experiments were shown. c, d Immortalized SVF from WT, Sqstm1–/–, and Sqstm1–/–Nbr1–/– mice were differentiated into brown adipocytes for Seahorse analyses. c Oxygen consumption rate (OCR) were determined (n = 6 biological replicates). Left: plot of OCR to time measured by Seahorse. Right: calculated ISO-stimulated respiration levels after subtracting levels of basal proton leak. d Extracellular acidification rate (ECAR) were measured (n = 6 biological replicates). Left: plot of ECAR to time measured by Seahorse. Right: calculated ISO-stimulated ECAR values after subtracting oligomycin-treated levels. Data are presented as mean ± SEM (a–d). *p < 0.05, **/##p < 0.01, ***p < 0.001, ****/####p < 0.0001. Two-tailed Student’s T-test (a, c, d bar graphs). Two-way ANOVA (b). Source data are provided as a Source Data file.

Fig 3: p62 and NBR1 interact with PPARγ in brown adipocytes.a Representative immunoblotting of p62 and NBR1 levels in cytoplasmic/nuclear fractions in primary brown adipocytes. Cells were treated with 0.5 µM isoproterenol (ISO) for indicated time. Densitometric quantification of nuclear NBR1 and p62 levels was also shown (n = 3 independent experiments). b, c Endogenous interaction of PPARγ with p62 and NBR1. p62 (b) or PPARγ (c) immunoprecipitates from nuclear lysates extracted from ISO and rosiglitazone-treated brown adipocytes were analyzed for the levels of specified proteins. Representative blots and densitometric quantification were shown (n = 3 independent experiments for both). d, e Recombinant FLAG-p62 (d) or FLAG-NBR1 (e) was incubated with GST and GST-PPARγ proteins separately and the interactions were analyzed by immunoblotting in glutathione-beads pull-down. Representative blots and densitometric quantification were shown (n = 4 independent experiments for d and n = 3 independent experiments for e). f HEK293T cells were transfected with cDNA vectors expressing WT/mutants of HA-p62 or HA-NBR1, and GST-PPARγ. The interacting proteins were pulled down using glutathione-beads against GST-PPARγ and analyzed by immunoblotting. Representative blots from three independent replicates with similar results were shown. Data are presented as mean ± SEM (a–e). *p < 0.05, **p < 0.01. Two-tailed Student’s T-test (a–e). Source data are provided as a Source Data file.

Fig 4: PARK2-dependent early degradation of VDAC1 is mediated through the ubiquitin-proteasome pathway. (a) HeLa cells stably expressing PARK2 were transiently transfected with si-NBR1 and si-SQSTM1 alone or in combination as indicated for 48 h, followed by vehicle or CCCP (10 µM) treatment for 3 or 6 h. Cells transfected with si-control were used as controls. Western blotting was performed to examine protein expression of NBR1, SQSTM1, and various mitochondrial proteins as indicated. (b) Densitometric analysis of VDAC1 after normalized to ACTB in (a) (mean±S.D., n=3). *P<0.05; NS, not significant. (c) PARK2 stably expressing HeLa cells was transiently transfected with HA-NBR1 and Flag-SQSTM1 alone or in combination as indicated for 24 h, followed by vehicle or CCCP (10 µM) treatment for 3 or 6 h. Cells transfected with empty vector were used as controls. Protein levels of NBR1 (using anti-NBR1 antibody), SQSTM1 (using anti-FLAG antibody), and various mitochondrial proteins were examined as described above. (d) Densitometric analysis of VDAC1 after normalized to ACTB in (c) (mean±S.D., n=3). *P<0.05; NS, not significant. (e) Regular HeLa cells were treated with CCCP (10 µM) for 3 h or 24 h. Western blot analysis was carried out to examine the expression of various mitochondrial proteins as indicated. (f) Densitometric analysis of protein levels after normalized to ACTB in (e) (mean±S.D., n=3). NS, not significant. (g) HeLa cells stably expressing PARK2 were treated with CCCP (10 µM) for indicated time periods in the presence or absence of lysosomal inhibitor bafilomycin A1 (BAF, 200 nM) or proteasome inhibitor lactacystin (LAC, 10 µM). Protein level of VDAC1 was examined by western blotting. (h) Densitometric analysis of protein levels after normalized to ACTB in (g) (mean±S.D., n=3). *P<0.05; NS, not significant

Fig 5: ULK2 is essential for skeletal muscle homeostasis. Our studies reveal that the Ulk2 gene presents a skeletal muscle–enriched pattern of expression in mice and that its deficiency in skeletal muscle, despite not impairing autophagy flux and proteolytic activities of the lysosome and proteasome, leads to robust accumulation of insoluble ubiquitinated protein aggregates associated with the adaptors p62 and NBR1. These findings suggest a key role for ULK2 in modulating the recognition and sequestration of ubiquitinated protein aggregates for degradation by autophagy (and potentially by the proteasome). The ensuing inability of ULK2-deficient muscle fibers to clear proteotoxic aggregates leads to atrophy, impaired force production, myofiber degeneration, and a generally unhealthy morphology of the muscle. Of note, these cellular events and functional outcomes are not observed in ULK1-deficient muscle. Autophagy is depicted with a phagophore and an autophagosome (white) and lysosome (blue).