Fig. 3

Bilirubin downregulates HMGCR expression in liver and human hepatocytes by promoting the ubiquitination and degradation of HMGCR. A Western blot analysis of hepatic HMGCR expression in ApoE^−/− mice treated with bilirubin (n = 3) or vehicle (n = 3). B Human normal hepatocytes (LO2 or hepatoma cell line (HepG2) were treated with different concentrations of bilirubin (BIL, 0, 3, 6, and 12 μM) for 24 h and the mRNA expression of HMGCR was determined by RT-qPCR. C LO2 or HepG2 cells were treated with different concentrations of bilirubin (0, 3, 6, and 12 μM) for 24 h and the protein level of HMGCR was measured by Western blot. D LO2 or HepG2 cells were treated with 12 μM of bilirubin for 0, 6, 12, or 24 h and the protein level of HMGCR was measured by Western blot. E HepG2 or LO2 cells were incubated with BIL (12 μM) or vehicle in the presence of 50 μg/ml of cycloheximide for the indicated time and the protein level of HMGCR was measured by Western blot. F HepG2 cells were treated with or without BIL (12 μM) in the presence of protease inhibitor MG132 (10 μM) for 24 h. Cell lysates were immunoprecipitated with anti-HMGCR antibody and then probed with an antibody against ubiquitin or HMGCR, respectively. G LO2 or HepG2 cells were incubated with or without BIL (12 μM) in the presence or absence of protease inhibitor bortezomib (Btz, 50 nM) for 24 h. The expression of HMGCR was determined by Western blot. Representative western blot images of three independent experiments (n = 3) with similar results are shown. Asterisks indicate the correct HMGCR bands. All the bands were quantified and the expression of HMGCR was normalized to GAPDH. Normalized values are below the bands