Fig.3. Requirement of DNA binding for DEC1 to repress CYP3A4 promoter.

(A) DEC1 repressed CYP3A4 promoter in a dose-dependent manner. HepG2 cells were cultured in 48-well plates and transfected with CYP3A4-DP-Luc (50 ng), hPXR (50 ng), corresponding concentration of DEC1 ( 1, 5, 10, 20, 50 ng ) and 5 ng of Null-Renilla reniformis luciferase plasmid. Vector plasmid was used to equalize the mount of plasmid DNA for each transfection. After 24 h incubation, the transfected cells were treated with Rif (10 μM) for another 24 h, and then, the cells were collected and analyzed for luciferase activity. (B) Diagrammatic presentation of a series of wild type and various mutated DEC1 constructs. (C) Differential effect of wild type and various mutated DEC1 on CYP3A4 promoter. HepG2 cells were cultured in 48-well plates and transfected with CYP3A4-DP-Luc (50 ng), hPXR (50 ng), wild type DEC1 or corresponding mutated DEC1 (50 ng) along with 5 ng of Null-Renilla reniformis luciferase plasmid for 24 h, and the transfected cells were treated with Rif (10 μM) for another 24 h, and then, the cells were collected and analyzed for luciferase activity. The data were expressed as normalized luciferase activity (based on Null-Renilla reniformis luminescence signal) (n = 3). All experiments were repeated at least three times, and data were expressed as mean ±SD. # p< 0.05, significantly difference from DEC1-transfected cells and vector-transfected cells.