FIG. 5.

Inhibition of ABCG5/8 in HepG2 cells increases phytosterol accumulation and down-regulation of NR0B2/SHP. HepG2 cells were incubated with LXR antagonist GSK2033 for 4 hours followed by addition of +/− GW4064 or +/− stig+sito overnight; cells were harvested and mRNA analyzed. (A) NR0B2/SHP. ^#P < 0.05 versus all conditions except stig+sito + GSK2033+GW4064. (B) Immunoblot analysis and quantification of SHP protein in hepatic lysates extracted from HepG2 cells incubated as describe in (A). ^#P < 0.05 versus all groups except GW4064 + stig+sito. SHP protein was normalized to GRB2 and expressed relative to untreated control. (C) LC/MS quantification of stigmasterol concentrations in HepG2 cells treated as described in (A). ^#P < 0.0001 versus all other groups. ⁺P < 0.0001 versus all other groups. (D) LC/MS quantification of sitosterol in HepG2 cells treated as described in (A). ^#P < 0.0001 versus all other groups. *P < 0.05 versus all groups except untreated. (E) ChIP assay demonstrating FXR binding to the promoter of NR0B2/SHP in cell homogenate from HepG2 cells incubated as described in (A). Data are presented as fold change over IgG. For all of these experiments, statistical analysis was performed by one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, ***P < 0.001, and ****P < 0.0001. Abbreviation: GRB2, growth factor receptor bound protein 2.