Fig. 5.

3β-Sulfated progesterone-based compounds modulate FXR activity. (A) Huh7 cells were transfected with the human expression constructs for RXR, FXRα2 / empty vector, FXR-luciferase reporter, and renilla construct for 24 hours. Fifty μM of compound or 1 μM GW4064 was used to treat the transfected cells for 24 hours. *P < 0.05 for treatment group versus vehicle control. RLU, relative light units. n = 3 ± SEM. Dose response curves of 0-100 μM EPAS and EPS, which were identified in the compound screen. RLU, relative light units. n = 3 ± SEM. (B) EPAS and EPS are unable to recruit the LxxLL motif to the FXR-LBD. GST-FXR-LBD and biotinylated LxxLL SRC-1 peptide were incubated in the presence of increasing EPAS, EPS, and CDCA concentrations and HTRF measured after 1-hour incubation at room temperature with orbital shaking. n = 3 ± SD. (C) Huh7 cells were transfected as in (A) and cotreated with 100 μM CDCA and increasing doses of EPAS and EPS. n = 3 ± SEM. (D) EPAS and EPS inhibit ligand-activated FXR recruitment of SRC-1. HTRF experiments were performed as in (B). HTRF reaction mix was incubated in the presence of 9 or 100 μM CDCA and 0-600 μM EPAS/EPS. n = 3 ± SD.