FIG. 2.

Effects of GAS41 siRNA on p53-dependent p21 gene induction and cell cycle progression. (A) GAS41 siRNA-mediated GAS41 knockdown in U2OS cells. Cells were transfected in duplicate with the indicated siRNAs, and whole-cell lysates were analyzed for relative levels of GAS41 and BAF53. (B) GAS41 siRNA-mediated induction of the p21 gene in U2OS cells. Cells were transfected with the indicated siRNAs and, after 48 h, treated with actinomycin D (10 ng/ml) for 6 h as indicated. Total RNA was analyzed by RT-PCR to estimate the relative expression levels of the p21 and GAPDH mRNAs. Relative levels of p21 gene expression (normalized to GAPDH expression) are shown in italics. (C) Effects of GAS41 siRNA on the levels of GAS41 and p53 in U2OS cells. Cells were transfected with the indicated siRNAs and, after 48 h, treated with actinomycin D (10 ng/ml) for 6 h as indicated. Total cell lysates were analyzed by immunoblotting for GAS41, p53, and actin. (D) Effects of GAS41 siRNA on cell cycle progression. U2OS cells were transfected in duplicate with the indicated siRNAs for 48 h and analyzed for DNA content by fluorescence-activated cell sorter analysis. Relative distributions of cell cycle were obtained from the analysis of FlowJo software. (E) Failure of GAS41 siRNA to induce p21 gene expression in p53-deficient H1299 cells. H1299 cells were transfected with control or GAS41 siRNA and, after 24 h, with empty vector (1 μg [−]) or vector expressing p53 (1 [+] or 2 [++] μg) as indicated. Total RNA was analyzed by RT-PCR for p21 and GAPDH mRNAs. (F) Effects of GAS41 siRNA on the levels of GAS41 and ectopically expressed p53 in H1299 cells. Cells were transfected with control or GAS41 siRNA and, after 24 h, with empty vector (1 μg [−]) or vector expressing p53 (1 [+] or 2 [++] μg). After 48 h, total cell lysates were analyzed by immunoblotting for GAS41, p53, and actin.