Figure 4

Simvastatin-induced p27 upregulation was Skp2-dependent and promoted G0/G1 phase cell cycle arrest in HepG2 cells. (a) Simvastatin decreased Skp2 protein expression in HepG2 cells. HepG2 cells were treated with simvastatin (0, 5, 10 or 20 μg/ml) for 24 h, and then cell lysates were harvested to detect the protein expression levels of Skp2 and β-actin by immunoblotting. (b) Simvastatin inhibited Skp2 mRNA expression in HepG2 cells. After the same treatment, cells were collected for analysis of Skp2 and GAPDH mRNA expression levels by RT-PCR and real-time PCR. (c) Simvastatin reduced Skp2 promoter activity. HepG2 cells were transfected with a pGL4.18-Skp2 promoter plasmid for 24 h and were then treated with simvastatin (0 or 20 μg/ml) for 24 h. The cell lysates were harvested to assay luciferase activity using a dual-luciferase assay kit. Data were normalized to Renilla luciferase activity and expressed as fold inductions of the control. (d) Skp2 overexpression rescued cells from simvastatin-induced G0/G1 cell cycle arrest. Control and Skp2-overexpressing HepG2 cells were treated with simvastatin (0, 10 or 20 μg/ml) for 48 h, and then the cells were collected for DNA content assay by flow cytometry. (e) Simvastatin was unable to change Skp2 and p27 protein expression levels in Skp2-overexpressing HepG2 cells. Control and Skp2-overexpressing HepG2 cells were treated with simvastatin (0 or 20 μg/ml) for 24 h, and then the cell lysates were collected for protein expression detection by immunoblotting using Skp2, p27 and β-actin antibodies. The results were obtained from three independent experiments. Data are expressed as the mean±S.E.M. of three independent experiments. **P<0.01, ***P<0.001