FIGURE 6.

FOXO1 mediates the AMPK-induced up-regulation of ORP150. A and B, HepG2 cells were transfected with FOXO1 siRNA (FOXO1 si) or control siRNA (Ctr si) and then treated with AICAR (1 mm) for 24 h. A, ORP150 mRNA was measured by quantitative RT-PCR, and results are expressed as -fold of control. *p < 0.05. The effectiveness of FOXO1 knockdown was examined by anti-FOXO1 antibody (upper panel). B, ORP150 protein expression was analyzed by Western blot. Representative data and quantitative analysis from three independent experiments are shown. *, p < 0.05. C, a representative Western blot shows the relative levels of endogenous and ectopic FOXO1. FOXO1 Thr-24 phosphorylation, total FOXO1 in hepatocytes infected with FOXO1-ADA, and FOXO1-Δ256 expression vectors are shown. The Δ256 form of FOXO1 lacks the epitope recognized by the FOXO1 antibody. D, overexpression of FOXO1 increased ORP150 protein levels. HepG2 cells were transfected with FOXO1-ADA or FOXO1-Δ256 in the absence or presence of AICAR (1 mm). The protein expression of ORP150 was examined. Representative blots from three independent experiments are shown. HepG2 cells transfected with FOXO1-ADA or FOXO1-Δ256 were treated with AICAR (1 mm) for 24 h (E) or HepG2 cells were incubated with AICAR (0–2 mm) for 24 h (F). After treatment, the protein-DNA complex was immunoprecipitated with an anti-FOXO1 antibody. The FOXO binding site in the DNA-protein complex was amplified by PCR. Representative blots from three independent experiments are shown.