Figure 5

STAT3C mutants maintained Skp2 expression to prevent p27 accumulation and G0/G1 cell cycle arrest in simvastatin-treated HepG2 cells. (a) Simvastatin inhibited the Jak1/Jak2-STAT3 pathway in HCC cells. HepG2 cells were treated with simvastatin (0, 5, 10 or 20 μg/ml) for 24 h, and then the cell lysates were harvested for the detection of protein levels by immunoblotting using p-Jak1, Jak1, p-Jak2, Jak2, p-STAT3, STAT3 and β-actin antibodies. (b) STAT3C mutants prevented simvastatin-induced G0/G1 cell cycle arrest in HepG2 cells. HepG2 cells were stably transfected with control vectors or STAT3C mutants and were then treated with simvastatin (0, 10 or 20 μg/ml) for 48 h. Then, the cells were collected for DNA content assay by flow cytometry. (c) Constitutive STAT3 activation in HepG2 cells upregulated Skp2 expression at the transcriptional level. Control and constitutive STAT3 activity levels in HepG2 cells were analyzed by RT-PCR to determine HepG2 Skp2 mRNA expression levels. (d) HepG2 cells stably expressing STAT3C maintained higher Skp2 levels and lower p27 levels. Control and STAT3C-expressing HepG2 cells were treated with simvastatin (0 or 20 μg/ml). Twenty-four hours later, the cell lysates were collected to detect protein expression by immunoblotting using p-STAT3, STAT3, 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<0.001