Figure 6. Bmal1 regulates cholesterol excretion to bile by modulating Gata4 expression.

(a) Wild-type primary hepatocytes were transfected in triplicate with siControl or siBmal1 and different mRNA levels were measured after 48 h (left). In a separate study, hepatocytes were transfected and used to detect protein levels (right). (b) Cholesterol efflux to bile acid acceptors was measured in wild-type hepatocytes transfected with siControl or siBmal1 (left). Also, primary hepatocytes were transfected with siControl or siGata4 (right). After 48 h, cells were labelled with ³H-cholesterol, washed and incubated with media containing 10 mM TUDC for 60 min. Radioactivity was determined by scintillation counting. (c) Wild-type hepatocytes were transfected with siGata4 or siControl and mRNA (left) and protein (right) levels of different indicated genes were quantified. (d) Primary hepatocytes were transfected with siControl, siGata4, siBmal1 or siBmal1+siGata4. After 48 h, mRNA (left) and protein (right) levels of indicated proteins were quantified. (e) Wild-type primary hepatocytes were transfected with siControl or siBmal1. After 48 h, cells were subjected to 2 h serum shock. At indicated times; mRNA levels of different genes were measured and corrected with 18S rRNA. The values in one well were normalized to 1. Other values represent fold-change compared with 0 h. (f) Hepatocytes from L-Bmal1^−/−Apoe^−/− and L-Bmal1^fl/flApoe^−/− mice (3 months, male) were cultured and subjected to serum shock and changes in mRNA levels of indicated genes were quantified at indicated times. Data in a–d were tested using the unpaired Student's t-test. Data in e and f were examined by two-way ANOVA. Values are mean±s.d. *P<0.05, **P<0.01 and ***P<0.001. Error bars represent s.d.