Fig. 3.

P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots (a, c, j) are shown in Supplementary Fig. 8. k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b, g, h. *P < 0.05, **P < 0.01, and ***P < 0.005 were considered statistically significant