EGFR signaling does not affect metformin-induced hCAR deactivation. (A) Human hepatocytes (HL#69) were treated with 0.1% DMSO, PB (1 mM), CITCO (1 µM), EGF (100 and 250 ng/ml), or their combinations for 24 hours. CYP2B6 expression was analyzed by real-time PCR. (B) Effects of erlotinib (10 µM) on the expression of CYP2B6, and phosphorylation of EGFR (Tyr1068) were determined by real-time PCR and Western blot analysis in human hepatocytes (HL#65, #66, #76, or #77) as detailed in Materials and Methods. (C) Immunoblotting analysis of EGFR (Tyr1068), total EGFR, and β-actin was performed in human hepatocytes (HL#77 and #81) treated with MET (1 mM) for 0, 15 minutes, 1 hour, 4 hours, and 24 hours. CYP2B6 mRNA expression was measured in human hepatocytes (HL#80 and #82) treated for 24 hours with 0.1% DMSO, MET (0.1, 0.5, 1 mM), PB (1 mM), CITCO (1 µM), erlotinib (1, 5, 10 µM), or their combinations as indicated in D and E. Data represent the mean ± S.D. of three independent transfections (**P < 0.01).