Figure 2: EE2 and TNC activate PXR in a synergistic fashion.
HG5LN GAL4-PXR-LBD (a), HepG2-PXR 3A4-Luciferase (b), and LS174T-PXR 3A4-Luciferase (c) cells were exposed to different concentrations of SR12813, TNC and EE2 either alone or in combination. Assays were performed in quadruplicate in at least three independent experiments and data are expressed as mean (±s.e.m.). Red dashed lines represent the theoretical activation curves obtained for the additive combination of EE2 and TNC activities calculated using the Bliss independence model19. (d) RT-qPCR analysis of CYP3A4 mRNA expression in control (Ctrl) or PXR overexpressing LS174T cells treated 48 h by solvent (0.1% DMSO) or the indicated concentration of ligand. Results were obtained from three separate experiments performed in duplicates. Data are expressed as mean (±s.e.m.) compared with DMSO treated cells, ***P<0.001 **P<0.01 *P<0.05 (Student’s t-test) compared to LS-CTRL cells. (e) RT-qPCR analysis of CYP3A4 mRNA expression in primary cultures of human hepatocytes (three independent donors: HH399, HH404 and HH408) treated 48 h by solvent (0.1% DMSO) or the indicated concentration of ligand. Results were obtained from experiments performed in triplicates. Data are expressed as mean (±s.d.) compared to DMSO treated cells, *P<0.05 (Student’s t-test). (f) Quantifications of CYP3A4 and GAPDH protein expression by western-blot (single experiment, upper panel) and CYP3A4 enzymatic activity (lower panel) in primary culture of human hepatocytes (HH408) treated 72 h by solvent (0.1% DMSO) or 1 μM ligand. Results for enzymatic activity were obtained from one experiment performed in triplicates. Data are expressed as CYP3A4/CellTiter Glow activities ratio as mean (±s.d.), **P<0.005 (Student’s t-test) compared with DMSO treated cells.
