FIG. 3.

Induction of CYP3A4 and HCE2 as a function of PXR. (A) Effect of siPXR on the induction of CYP3A4 and HCE2. Huh7 cells in 6-well plates were transiently transfected with a mixture containing 800 ng of siPXR or the corresponding vector (per well). After 72 h incubation, the transfected cells were treated with 8-MOP (25μM), RIF (25μM), or the same volume of DMSO (0.1%) for 30 h. Total RNA was isolated and analyzed for the expression of CYP3A4, HCE2, PXR, and GAPDH by RT-PCR. The PCR cycles were 35 for CYP3A4, 30 for HCE2, 32 for PXR, and 22 for GAPDH. Three independent experiments were performed, and the data are expressed as mean ± SD. *Significantly different from vector-transfected cells according to t-test (p < 0.05). (B) Effect of PXR overexpression on the induction of CYP3A4 and HCE2. Huh7 cells in 6-well plates were transiently transfected with a mixture containing 800 ng (per well) of PXR construct or the corresponding vector. After 12 h incubation, the transfected cells were treated with 8-MOP (25–50μM) or the same volume of DMSO (0.1%) for 72 h. Total RNA or cell lysates were isolated and analyzed for the expression of CYP3A4, HCE2, PXR, and GAPDH by RT-PCR. The PCR cycles were 29 for CYP3A4, and Western analysis was performed with 30 μg protein. (C) Enzymatic activity of CYP3A4 and HCE2. Huh7 cells were treated with DMSO or 8-MOP (50μM) for 48 h. Microsomes were prepared by differential centrifugation and assayed for the activities of CYP3A4 and HCE2 as described in the Materials and Methods. *Significantly different from vector-transfected cells according to t-test (p < 0.05).