Fig. 3.

Dexamethasone (DEX) inhibits transcription of Ppargc1a alternative promoter during brown adipocyte differentiation. (a–f) Mouse embryonic fibroblasts (MEFs) were isolated from wildtype C57BL/6J mice and induced for brown adipocyte differentiation. In brown adipogenic induction, MEFs were treated with DEX (n = 4). (a) Oil-Red O staining in differentiated mature brown adipocytes at 5 days (n = 4). Scale bar: 50 μm. (b) Immunoblotting measurement of adipogenic and thermogenic proteins in differentiated brown adipocytes, including GR, PGC-1a, UCP-1, PRDM16, VDAC, and cytochrome c (Cyto-C). β-tubulin was used as a loading control (n = 4). (c) Heatmap displaying brown adipogenic and thermogenic gene expression in differentiated brown adipocytes (vehicle vs. 10 μmol/L DEX). The mRNA expression was normalized to 18S rRNA (n = 3). PAN: gene expressions which are shared in brown and white adipocytes, WAT: white adipose tissue. (d) Mitochondrial DNA (mtDNA) copy number in differentiated brown adipocytes (vehicle vs. 10 μmol/L DEX). Mitochondrial gene expression was normalized to 18S rRNA and GAPDH (n = 4). (e) Ppargc1a mRNA expression transcribed from the canonical promoter (CP) and alternative promoter (AP) during brown adipocyte differentiation (n = 4). (f) Ppargc1a transcriptions from AP and CP in differentiated brown adipocytes treated with vehicle or 10 μmol/L DEX. Expression was normalized to 18S rRNA (n = 4). (g–j) MEFs were transfected with plasmids with scrambled or open reading frame of Ppargc1a transcribed from the AP for over-expression (OE) (AP-PGC1a OE) followed with brown adipogenic induction. (g) Ppargc1a transcription from AP and CP in MEFs transfected with scrambled or AP-PGC1a plasmids. Expression was normalized to 18S rRNA (n = 4). (h) Immunoblotting measurement of PGC-1a total protein content in MEFs transfected with scrambled or AP-PGC1a plasmids. β-actin was used as a loading control. (i and j) MEFs transfected with scrambled and AP-PGC1a OE plasmids were also treated with vehicle or 10 μmol/L DEX in brown adipocyte differentiation (n = 4). On day 5, differentiated brown adipocytes were stained by Oil-Red O (scale bar: 50 μm) (i), and contents of brown thermogenic and mitochondrial biomass proteins were measured using immunoblotting. β-actin was used as a loading control (n = 4). Data are representative of three separate experiments. Data are mean ± SEM and each dot represents one replicate; *P < 0.05, **P < 0.01, and ***P < 0.001; unpaired Student’s t-test with two-tailed distribution was used in panel (c–g) data analyses.