Figure 1.
Schematic illustration of chimeric hPXR constructs designed in the present study.
CMV promoter = cytomegalovirus immediate-early enhancer/promoter region; SV40 late polyA = simian virus 40 late polyadenylation signal.
Table 1.
Primers’ sequence used for qPCR.
Figure 2.
Activation of CYP3A4.XREM.luc luciferase reporter by various hPXR constructs in WT C3A and HEK293T cells.
The empty vector contained the pCI-neo plasmid instead of hPXR construct. The ligand (black columns) was 20 µM RIF while vehicle (white columns) was 0.02% (v/v) DMSO. The ratio of reporter luciferase activity to control renilla luciferase activity is indicated. Results are expressed as the mean±S.D. of normalized luciferase activity of three independent measurements. Statistical significance p<0.05: *, RIF-treated vs. respective DMSO control; ***, chimeric hPXR vs. WT hPXR vector.
Figure 3.
CYP3A4 mRNA expression in C3A cells transiently transfected with different hPXR constructs.
The relative mRNA levels compared with mRNA levels in the WT C3A without the addition of RIF control cells (relative expression value set to 1) were defined by the 2−ΔΔCT method. Results are expressed as mean±S.D., n = 3. Statistical significance p<0.05: *, RIF-treated vs. respective DMSO control; **, WT hPXR vs. empty vector; ***, chimeric hPXR vs. WT hPXR vector.
Figure 4.
C3A stably transfected with chimeric hPXR-p53-AD construct (A5) has improved CYP3A4 expression at gene, protein and activity levels.
(A) CYP3A4 mRNA expression in WT C3A and A5. The relative mRNA levels compared with mRNA levels in the WT C3A without the addition of RIF control cells (relative expression value set to 1) were defined by the 2−ΔΔCT method. (B) The expression of CYP3A4 protein increased in A5 cells compared to WT C3A cells. Total proteins were isolated from WT C3A and A5 cells and analyzed by Western blotting. β-actin was used as a gel loading control. (C) CYP3A4 catalytic activity was determined by monitoring the 6β-hydroxylation of CYP3A4-dependent testosterone in microsomes isolated from A5 or WT C3A cells, as measured by HPLC. (D) CYP3A4 catalytic activity was determined by monitoring the formation of 6β-hydroxytestosterone in the medium of WT C3A or A5 cells, as measured by HPLC. For induction studies, 20 µM RIF was added 48 h before measurement. Data are expressed as mean±S.D. of three independent experiments. Statistical significance p<0.05: *, RIF-treated vs. respective DMSO control; **, A5 vs. WT C3A. N.D., not detected.