Fig. 5.

A, substitution of the IR1 element in the BSEP promoter. Multiple nucleotide substitutions were introduced in the IR1 element by site-directed mutagenesis, resulting in an IR1 mutant, pBSEP-IR1-Mut. Four and three nucleotides were mutated in the first half and the second half, respectively, to ensure complete destruction of the site. B, the IR1 element was required for maximal activation of BSEP promoter by 22(R)-OHC. Huh 7 cells were transfected with 100 ng of either pBSEP-Luc (pBSEP-IR1-wt) or the IR1 mutant (pBSEP-IR1-Mut) with 100 ng of FXR expression plasmid, and 10 ng of the null-Renilla luciferase plasmid. Sixteen hours post-transfection, cells were treated with 10 μM 22(R)-OHC for 30 h. Luciferase activities were measured by the Dual-Luciferase Reporter assay system.