Fig 1: The PRDM16-mediated repression of SREBP target promoters is dependent on SREBP1/2 activation. (A) HepG2 cells were transfected with the HMG-CoA synthase promoter-luciferase construct (SYNSRE) together with GFP or PRDM16 (P16) cDNA. Twenty-four hours after transfection, the cells were placed in lipoprotein-deficient media. Where indicated, media was supplemented with 25-hydroxycholesterol (25-HC) to block SREBP1/2 activation. Forty-eight hours after transfection, the 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 and treated as in (A). 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 duplicate. 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, and *** p < 0.001. ns, not significant.