G9a recruitment to the CYP7A1 promoter and subsequent H3K9 methylation after bile acid treatment is dependent on SHP expression. (A and B) HepG2 cells were transiently transfected with either pSUPER or pSUPER-siSHP and treated with 50 μM CDCA for 18 h. (A) Levels of mRNA were determined by real-time RT-PCR relative to 36B4 mRNA levels. The standard error of the mean was calculated from triplicate samples, and statistical significance was determined by a Student's t test. , P < 0.01. (B) ChIP assays were performed using antibodies against SHP and G9a. Precipitated chromatin was extensively washed and analyzed as described in the legend of Fig. 4E. (C) HepG2 cells were infected with Ad-empty or Ad-siSHP virus and treated with 50 μM CDCA or vehicle for 18 h. Cells were then collected for ChIP assays using antibodies against SHP, G9a, diacetylated H3K9/K14, or dimethylated H3K9, or using normal serum (NS). Precipitated chromatin was extensively washed and analyzed as described in the legend of Fig. 4E. di-AcH3 K9/K14, diacetylated H3 K9/K14; di-Met H3K9, dimethylated H3K9; hCYP7A1, human CYP7A1; hGAPDH, human GAPDH.