FIG. 4.

Decreased expression of ABCG5/8 enhances the inhibitory effect of phytosterols on FXR target gene expression. HepG2 cells were transfected with ABCG8 siRNA or nontargeting siRNA for 24 hours followed by addition of +/− GW4064 or +/− stig+sito overnight, after which cells were harvested and mRNA analysis was performed. (A) ABCG8. ^#P < 0.0001 versus all groups except siRNA control. ^$P < 0.0001 versus all groups except untreated control. (B) NR0B2/SHP. ^#P < 0.05 versus all groups except ⁺ and ^$. ⁺P < 0.05 versus all groups except ⁺ and ^#. ^$P < 0.05 versus all groups except ^# and ⁺. ^&P < 0.05 versus all other groups. (C) HepG2 cells were incubated with IL-1β (to reduce ABCG5/8 expression) for 4 hours followed by GW4064 +/− stig+sito overnight, after which cells were harvested and mRNA analysis of NR0B2/SHP was performed. ^#P < 0.05 versus all groups except GW4064 + stig+sito + IL-1β. (D) Primary mouse hepatocytes (Mouse 1°) were incubated as in (C), and Nr0b2/SHP mRNA was analyzed. ^#P < 0.01 versus all groups except stig+sito + IL-1β + GW4064. (E) Abcb11 in primary mouse hepatocytes. ^#P < 0.001 versus all groups except GW4064 + stig+sito + IL-1β. (F) Huh7 cells were incubated as in (C), and ABCB11 mRNA was analyzed. ^#P < 0.0001 versus all other groups. ⁺P < 0.0001 versus all other groups. ^$P < 0.05 versus all other groups. ^&P < 0.05 versus all other groups. Gene expression was determined after normalization to HPRT1 relative to results obtained from untreated controls. For all of these experiments, statistical analysis was performed by one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.