Fig. 4
β-Adrenergic signal is required for the induction of beige-selective genes mediated by PPARγ ligand. a WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs were differentiated for beige adipogenesis in the presence or absence of propranolol (Pro), as schematically illustrated (top), and ORO staining was performed (bottom). b Whole-cell lysates (WCL) from WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs differentiated under Pro (100 nM) plus or minus condition were subjected to immunoprecipitation (IP) with anti-V5 antibody, followed by immunoblot (IB) analysis with anti-P-JMJD1A (pSer265) antibody. c qPCR analysis of beige-selective genes and general adipogenic genes in WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs under Pro plus or minus condition (mean ± s.e.m. of three technical replicates). d Immunoblotting with anti-UCP1 or anti-total OXPHOS antibodies cocktail using WCL from indicated viral transduced im-scWATs, differentiated under Pro plus or minus condition. e OCRs (basal, maximum, and uncoupled) of WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs, differentiated under Pro plus or minus condition. Data are mean ± s.e.m. of five technical replicates. f, g Immunoblotting with anti-PRDM16, anti-P-JMJD1A, anti-V5, anti-PPARγ, or anti-PGC1α antibody following immunoprecipitation with anti-PRDM16 antibody (f) or with anti-V5 antibody for JMJD1A (g), from WCL of differentiated WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs. Uncropped images of the blots (b, d, f, g) are shown in Supplementary Fig. 8. Analysis of variance was performed, followed by Tukey’s post hoc comparison in e. *P < 0.05, **P < 0.01, and ***P < 0.005 were considered statistically significant. h Schematic drawing of p265-JMJD1A-PPARγ-PGC1α-PRDM16 protein complex. Integration of β-adrenergic-cAMP signaling and the PPARγ ligand binding is mediated by JMJD1A through a mechanism where pS265-JMJD1A forms a complex with PGC1α, PRDM16, and PPARγ to mediate expressions of beige-selective genes
