Fig 1: PRDM16 targets the nuclear forms of SREBP1a, SREBP1c, and SREBP2. (A) HepG2 cells were transfected with the HMG-CoA synthase promoter-luciferase construct (SYNSRE) together with GFP or PRDM16 (P16) cDNA in the absence (EV) or presence of cDNA encoding the nuclear forms of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. (B) HepG2 cells were transfected with the HMG-CoA synthase promoter-luciferase construct (SYNSRE) together with non-targeted (C) or PRDM16 (P16) shRNA in the absence (EV) or presence of cDNA encoding the nuclear forms of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. EV, empty vector. (C) HepG2 cells were transfected with a minimal promoter-luciferase construct containing a single binding site for the yeast transcription factor Gal4 (G1E1B-Luc) together with expression vectors for the DNA binding domain of Gal4 (Gal4), or the same DNA binding domain fused to the nuclear forms of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2), and either GFP or PRDM16 (P16). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. (D). HepG2 cells were transfected with the same promoter-reporter construct as in (A) together with the DNA binding domain of Gal4 (Gal4), or the same DNA binding domain fused to the transactivation domain (TAD) of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2), and either GFP or PRDM16 (P16) cDNA. Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. Cells were also transfected with the β-galactosidase gene as an internal control for transfection efficiency. Luciferase values (relative light units, RLU) were calculated by dividing the luciferase activity by the β-galactosidase activity. The data represent the average −/+ SEM of at least three independent experiments performed in duplicates. The RLU of WT promoter-reporter constructs transfected with either GFP or non-targeted shRNA were set to 1. p-values lower than 0.05 were considered statistically significant. * p < 0.05, ** p < 0.01, and **** p < 0.0001.