Fig 4. Effects of various compounds on transactivation of rat CAR3.

HepG2 cells were transfected with rat CAR3 or empty expression vector and the CYP2B6 promoter and enhancer linked to a luciferase reporter vector (A) or luciferase reporter vector with CYP3A4 proximal and distal promoter regions (B) before seeding in 96-well plates as described in Materials and Methods. Cells were treated with the same panel of chemicals for 48 h before detection of fluorescence and luminescence. All luminescence values were normalized for cell viability. Data represents the mean ± SE from three independent experiments in triplicate expressed as fold activation above vehicle control treated cells. All data is normalized against fold activation values from transactivation assays with an empty expression vector. An asterisk denotes significant difference from vehicle controls at a level of p < 0.01. (C-D), Representative clotrimazole dose-response curves for rCAR3/2B6 (C) and rCAR3/3A4 (D). Cells in triplicate wells were treated with 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 20 μM clotrimazole (C) or 0.1, 0.5, 1, 5, 10 and 20 μM clotrimazole (D) for 48 h before luminescence was detected and normalized against cell viability. Results are expressed as fold activation above 0.1% DMSO treated cells. Data represent the mean ± SE from three independent experiments triplicate. EC₅₀ and E_MAX values were calculated using nonlinear regression of a typical log dose-response curve.