FIGURE 1.
SKI negatively regulates p53. A, overexpression of SKI leads to a decrease of p53. p53, p21waf1 level was examined with immunoblot analysis in mink lung epithelial cells overexpressing SKI (Ski13) or vector control. B, half-life of p53 was decreased in Ski13 cells. C, SKI enhances decrease of p53 by MDM2 but not by mutant MDM2. Various expression plasmids were introduced into HCT116 cells without Tp53, and the p53 was examined by immunoblot (IB) analysis with anti-p53 antibody. The mutant MDM2 has alanine at amino acid 464 instead of cysteine (C464A). The amount of plasmids was made equal by adding vector DNA, and the transfection efficiency was monitored with GFP expression. MDM2 and SKI expression were monitored with anti-HA and anti-FLAG antibodies. CHX, cycloheximide. D, reduction of SKI results in an increase of p53 level. siRNA for SKI was introduced into LNCaP cells, and the levels of SKI, p53 and GAPDH were examined by immunoblot analysis. E and F, reduction of SKI results in a decrease of cell number and an increase of apoptosis. 48 h after siRNA for SKI was introduced into LNCaP cells, the cell number was measured using CellTiter Glo (E), and the sub-G1 content was measured with FACS analysis (F). G, reduction of SKI results in an increase of several p53 target genes. SYBR quantitative PCR was used to measure the quantity of transcript level of p53 target genes in cells with transfection of control siRNA or siRNA for SKI. The level in control cells was arbitrarily set as 1 followed by correction with relative total RNA level using GAPDH measurements.