Figure 2

Simvastatin-induced p21 and p27 protein upregulation was associated with transcriptional activation and protein degradation inhibition, respectively. (a) Simvastatin modulated p21 mRNA expression. HepG2 cells were treated with simvastatin (0, 5, 10 or 20 μg/ml) for 24 h, and p21, p27 and GAPDH mRNA expression levels were detected by RT-PCR and real-time PCR. (b) The effects of p21 and p27 protein stability in simvastatin-treated HCC cells. HepG2 cells were treated with 10 μg/ml CHX alone for 1, 2, 4, 6 or 12 h or 10 μg/ml simvastatin for 12 h. After 12 h, simvastatin-treated cells were co-treated with 10 μg/ml CHX for 1, 2, 4, 6 or 12 h. The cell lysates were harvested to detect p21, p27 and β-actin protein expression by immunoblotting. The intensity of each protein signal was determined by ImageJ software (downloaded from the NIH website (http://rsb.info.nih.gov/ij)). (c) Inhibition of proteasomal degradation promoted p21 accumulation, but not p27 accumulation, in simvastatin-treated HCC cells. HepG2 cells were treated with 20 μg/ml simvastatin with or without 10 μM MG132 for 24 h and then subjected to immunoblotting for the detection of p21, p27 and β-actin expression levels. Data are expressed as the mean±S.E.M. of three independent experiments. Statistically significant differences between the un-treated and treated groups are indicated. **P<0.01, ***P<0.001