Figure 8.

p21 knockdown rescued the down-regulation of Rb phosphorylation and replicative senescence induced by PDCD4 knockdown. (A) p21 knockdown suppressed the down-regulation of Rb phosphorylation induced by PDCD4 knockdown. HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, each cell sample was then treated with negative control siRNA (nc), PDCD4-specific p2 siRNA (p2, Upper) or k603 siRNA (k603, Lower). The cells were then cultured for a further 24, 48, or 72 h and then subjected to a Western blotting analysis using anti-p-Rb (780), anti-p-Rb (807/811), anti-p21, anti-PDCD4 and anti-β-actin antibodies. (B) p21 knockdown up-regulated Rb-phosphorylation. HepG2 cells were treated with negative control siRNA (nc) or p21-specific siRNA (p21) and subjected to a Western blotting analysis with anti-p-Rb (780), anti-p-Rb (807/811), anti-p21 and anti-β-actin antibodies after culturing for the times indicated in the figures. (C) p21 knockdown suppressed the PDCD4 knockdown-induced replicative senescence. (Left panel) HepG2 cells were first treated with negative control siRNA (nc) or p21-specific siRNA (p21). After culturing for 24 h, both the nc and p21 knockdown cells were again treated with either negative control siRNA (nc-nc and p21-nc) or PDCD4-specific p2 siRNA (nc-p2 and p21-p2). (Right panel) HepG2 cells were treated the same as in the left panel using PDCD4-specific k603 siRNA. The cells were then fixed and stained with the β-galactosidase assay kit after culturing for an additional 96 h. The galactosidase-positive cell numbers represent the average cell number of three different fields. Thousands of cells were counted in each field. The experiments were repeated twice, and similar results were obtained each time. t-test: *p < 0.05; **p < 0.005.