(A) Oil-red-O staining of mature adipocytes (day 6) from PPARγ-deficient cells expressing retroviral PPARγ2 and either retroviral- PRDM16 or vector control (ctl). These adipocyte cultures were analyzed by real-time PCR for their expression of: differentiation markers common to WAT and BAT (A); brown fat cell-selective genes (as indicated) (B); brown fat- thermogenic genes (UCP1, deiodinase-d2 (dio-2), PGC-1α) with and without cAMP treatment (C); mitochondrial components (as indicated) (D); and white fat cell-selective markers (psat1, serpin3ak, resistin) (E). (n=3, mean ± SD). * p < 0.05; ** p < 0.01

(A) Immunohistochemistry for cidea protein expression in endogenous WAT and BAT, and in ectopic subcutaneous fat pads formed from fibroblasts expressing PPARγ2 and either vector control (ctl) or PRDM16. (B–D) 8-weeks after transplantation, ectopic fat pads were analyzed by real-time PCR for expression of: PRDM16 (B); differentiation markers common to white and brown fat (PPARγ and adiponectin) (C); brown fat-selective genes (UCP1, cidea, PGC-1α, elovl3, PPAR-α, endogenous PRDM16 (-3′UTR) and the white fat selective marker, resistin (D). (n=10 mice per cell line, mean ± SE). * p < 0.05; ** p < 0.01

(A) PRDM16 mRNA levels in immortalized brown fat cells expressing shRNA targeted to PRDM16 or a scrambled (SCR) control- shRNA before (day 0) and after their differentiation (day 5) into adipocytes. (B) Gene expression in brown fat cells (day 5) expressing sh-PRDM16 or sh-SCR including: markers common to white and brown fat cells (aP2, PPARγ, adiponectin) and resistin, a white fat cell selective gene. (C) The differentiation-linked mRNA induction (day 0 to day 5) of brown fat- selective genes (as indicated) in sh-PRDM16 and sh-SCR expressing cells. (D–F) Gene expression in adipocytes (day 6) from sh-PRDM16 and sh-SCR expressing primary brown preadipocytes including mRNA levels of: PRDM16 (D); adiponectin, PPARγ and resistin (E); brown fat- selective genes (as indicated) (F). (G) Western blot analysis of UCP1 protein levels in primary brown fat cells expressing sh-PRDM16 or sh-SCR control with and without cAMP treatment. (H) mRNA levels of various mitochondrial components in adipocytes from sh-PRDM16 and sh-SCR expressing primary brown preadipocytes. (n= 3–5, mean ± SD). * p < 0.05; ** p < 0.01

(A) Real-time PCR analysis of PRDM16 mRNA expression in BAT relative to WAT; and its expression in the adipocyte fraction (Adip.) compared to the stromal vascular fraction (SV) of BAT (n= 6, mean ± SD). (B) PRDM16 mRNA expression in adipocytes from immortalized BAT cell lines (BAT1 and BAT2) and three WAT cell lines (3T3-F442A, 3T3-L1, C3H-10T1/2) (n=3–5 samples per cell line, mean ± SD). (C) PRDM16 mRNA levels during the differentiation of immortalized BAT cell lines (n=3 for each of 2 separate cell lines, mean ± SD). (D) Northern blot analysis of PRDM16 mRNA and control, 36B4 mRNA, in adult mouse tissues. (E) Expression of PRDM16, UCP1, PGC-1α and adiponectin in BAT after a 4°C cold exposure of mice for 4 hours (n=5 mice per group, mean ± SD) (rt: room temperature). * p < 0.05; ** p < 0.01.

(A) Representative transmission electron micrographs of fibroblasts and mature adipocytes (day 6) from PPARγ-deficient cells expressing retroviral PPARγ2 and either retroviral- PRDM16 or vector control (ctl). (B) Comparison of mitochondrial volume densities from cells depicted in (A) (n= >20 micrographs per group, mean ± SD). (C, D) Total mitochondrial oxygen consumption and uncoupled respiration in mature adipocytes expressing either PRDM16 or control vector under basal conditions (n=4, mean ± SD) (C), or after stimulation with a cAMP analog for 12 hours (n=4, mean ± SD) (D). L: lipid droplet. * p < 0.05; ** p < 0.01

(A) The transcriptional activity of the -2 kb region of PGC-1α in response to PRDM16 or vector control expression in brown fat preadipocytes (n=3, mean ± SD). (B) PGC-1α promoter activity in response to PRDM16 or vector expression in PGC-1α-deficient cells (n=3, mean ± SD). (C) The transcriptional activity of Gal4-DNA binding domain (DBD) fusion proteins containing PGC-1α, PGC-1β, or PRC in response to PRDM16 or vector expression (n=3, mean ± SD). (D) Flag-PRDM16 and its associated proteins were immunoprecipitated from brown fat preadipocytes and analyzed by Western blot to detect PGC-1α and PGC-1β (E) GST fusion proteins containing different regions of PGC-1α were incubated with ³⁵S- labeled PRDM16 protein. ERR-α was used to demonstrate binding to the 1–190 and 200–350 regions of PGC-1α. (F) PGC-1α and PRDM16 were co-precipitated from cos7 cells that had been transfected with HA-PGC-1α and either wildtype (WT) or R998Q mutant PRDM16. The input was 2% of the cell lysate used for immunoprecipitation. (G) WT or R998Q mutant PRDM16 were expressed with PPARγ2 in PPARγ^−/− fibroblasts. After differentiation into adipocytes (day 6), real-time PCR was used to measure the mRNA expression of: brown fat-selective genes (as indicated); and resistin, a white fat selective gene (n=3, mean ± SD). * p < 0.05; ** p < 0.01

(A) The fat-specific aP2 promoter/enhancer was used to express PRDM16 in WAT depots. Western blot analysis for PRDM16 protein expression in: non-transgenic, wildtype (wt) BAT; wt WAT; and WAT from two strains of aP2-PRDM16 transgenic mice (aP2-T1 and aP2-T2). POL-II protein expression was used to control for loading. (B) Expression of BAT-selective genes (as indicated) and resistin in WAT from wildtype (wt) and aP2-T1 transgenic mice. This gene set was also measured in WAT from wt, aP2-T1 and aP2-T2 mice that had been treated with CL 316, 243 (n= 7–10 mice per group, mean ± SE). (C) Immunohistochemistry for UCP1 protein (brown stain) in sections of WAT from wt and transgenic mice (T1 and T2) after treatment with CL 316, 243. * p < 0.05; ** p < 0.01