Figure 3. Proteasomal inhibition attenuates TCPOBOP-induced CAR transcriptional activation in HepG2 cells.
Cells were cotransfected 18/V5-His-mCAR or empty vector combined with either the luciferase reporter plasmid containing −1.8 kb upstream region of the CYP2B6 gene promoter (−1.8-kb-luc) (A) or the (NR1)5-tk-luciferase [(NR1)5-tk-luc] reporter plasmid (B). The Renilla construct (phRL-tk) was also co-transfected as a normalization control. Cells were then treated with DMSO (0.1% v/v), TCPOBOP (250 nM dissolved in 0.1% DMSO, final concentrations), MG132 (5 µM in 0.1 1% DMSO, final concentrations), or MG132 plus TCPOBOP, after which cells were harvested and luciferase activity measured. Luciferase activity of each sample was normalized to Renilla activity, and expressed as means ± SD of triplicate determinations. Results shown are representative of three independent experiments. *Significantly different (p<0.0001) compared to DMSO (+mCAR-V5); #significantly different (p<0.0001) compared to TCPOPOP (+mCAR-V5), based on one-way ANOVA with post-hoc Tukey multiple comparisons test.