6,7-Dimethylesculetin activates CAR in primary hepatocytes. (a) Structures of 6,7-dimethylesculetin and 4′-hydroxyacetophenone. (b) Primary hepatocytes from either WT (+/+) or CAR knockout animals (–/–) were cultured in William’s E medium (Invitrogen) and incubated with solvent (CO) or increasing concentrations of 4′-hydroxyacetophenone (HA; 10 μM, or 50 μM), or 6,7-dimethylescule μM) for 24 hours. Total cell RNA was prepared, and 15 μg of each RNA sample were used for Northern hybridization as indicated. (c) Primary hepatocytes were isolated from humanized CAR transgenic mice and cultured in the presence of either solvent or 10 μM or 50 μM 6,7-dimethylesculetin for 24 hours. Fifteen micrograms of total RNA from cells were analyzed by Northern hybridization. (d) Primary hepatocytes were isolated from either CAR knockout or humanized CAR transgenic mice and cultured in the presence of either solvent, or 50 μM 6,7-dimethylesculetin for 3 hours. Fifty micrograms of nuclear extract protein (upper panel) or 50 μg of cell total protein (lower panel) from each treatment were fractionated by SDS-PAGE and immunoblotted with an anti c-Myc antibody that recognizes the N-terminal epitope tag on the human CAR expressed in these animals.