FIG. 8.
p27Kip1 is involved in apoptin-induced apoptosis. (A) Downregulation of p27Kip1 protects against apoptin-induced apoptosis. PC3 cells were transfected with a GFP control or GFP-apoptin in the presence and absence of a p27Kip1-specific siRNA. (Left) Apoptosis was measured by flow cytometric detection of hypodiploid DNA at the indicated time points. The data represent the means from three independent experiments. (Right) The efficiency of p27Kip1 downregulation by the specific or scrambled control siRNA was investigated at 36 h posttransfection by immunoblotting with a p27Kip1-specific antibody. Tubulin served as a loading control. (B to D) Apoptin-induced apoptosis is inhibited in cells with suppressed expression of Skp2 and Cks1, two components of the p27 degradation machinery. (B) PC3 cells were transfected with either a control siRNA, two different Skp2 siRNAs (left panels), or two different Cks1 siRNAs (right panels). The protein levels of Skp2, Cks1, p27, and actin were analyzed by immunoblotting at 72 h post-siRNA transfection. (C) PC3 cells transfected with either control siRNA, Skp2 siRNA no. 2, or Cks1 siRNA no. 2 were treated with Tat-apoptin (1 μM) at 36 h posttransfection, and the percentage of apoptosis was measured after 24 h of further incubation. (D) Immortalized Skp2-deficient murine fibroblasts and the respective WT control cells were treated with Tat-apoptin, and the percentage of apoptosis was evaluated after 24 and 48 h of treatment. Data represent the averages of three independent experiments.