FIGURE 2.

Ski suppresses the transactivation ability of p53. A and B, Ski inhibits the induction of p21 and PIG3 transcription (A) and p21 protein expression (B) after ActD treatment. A, cells were treated with 1 nm ActD for the indicated time periods, and RNA was purified from each sample and analyzed by quantitative real-time PCR. Each value was normalized to the HPRT1 expression levels. B, cells were treated with 1 nm ActD for the indicated time periods, and the cell lysates were analyzed by immunoblotting with the indicated antibodies. C and D, Ski knockdown augments p21 and mdm2 mRNA (C) and p21 protein (D) expression levels after ActD treatment. C, MCF7 cells were transfected with the indicated siRNAs and treated with 10 nm ActD for 4 h. Quantitative real-time PCR analyses of the p21 and mdm2 mRNA levels in MCF7 cells as in A are shown. D, MCF7 cells were transfected with the indicated siRNAs and treated with 10 nm ActD for 4 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. E, Ski knockdown enhances cell cycle arrest after ActD treatment. MCF7 cells were transfected with the indicated siRNAs and treated with 10 nm ActD for 8 h. The cells were labeled with 10 mm BrdUrd for 1 h and then immunostained with an anti-BrdUrd antibody. The average percentage of BrdUrd-positive cells is shown. F, transactivation activity of p53 is increased upon Ski knockdown. Reporter gene constructs containing p53-responsive elements (pp53-TA-Luc) or the control vector alone (pTA-Luc) were co-transfected into MCF7 cells with the indicated siRNAs. After 24 h, the luciferase activity was measured. The experiment was performed in triplicate, and the data are represented as mean -fold activation ± S.D.