Fig. 6.

Chromatin immunoprecipitation assays of insulin effect on FoxO1, SREBP-1c and HNF4α recruitment to CYP7A1 chromatin. A. Human primary hepatocytes (HH1308) were treated with 10 nM insulin for the time indicated. Cells were harvested for ChIP assay as described under Materials and Methods. An antibody against HNF4α, SREBP-1 or FoxO1 was used to immunoprecipitate chromatins for PCR amplification and analysis. Ten % of cell lysate was used as input. Non-immune IgG was used as negative controls. B. ChIP assays of the effect of insulin and T0901317 on HNF4α recruitment of PGC-1α to CYP7A1 chromatin. HepG2 cells in 100 mm dish were transfected with HA-tagged PGC-1α (10 μg). Cells were then treated with insulin (100 nM) or T0901317 (1 μM) for 24 hr. Chromatins were precipitated with an antibody against HA tag or HNF4α for ChIP assays. Ten % of cell lysate was used as input. Non-immune IgG was used as negative controls. C. ChIP assays of HepG2 cells over-expressing SREBP-1c or FoxO1. HepG2 cells in 100 mm dish were transfected with 10 μg of HA-tagged PGC-1α, pcDNA3 empty vector, FoxO1 or SREBP-1c expression plasmid was over-expressed as indicated. Cells were harvested for ChIP assay as described under Experimental procedure. An antibody against HA-tag or HNF4α was used to immunoprecipitate chromatins for PCR amplification and analysis. Ten % of cell lysate was used as input. Non-immune IgG was used as negative controls.