Requirement of p21, but not p53, for inhibition of proliferation by LY. (a) Response of HCT116 colorectal cancer cells deficient for p21 to LY. Cells were treated with LY or, as a control, with vehicle (DMSO), and cell numbers were assessed. Measurements were performed in triplicates. (b) Response of p53^+/+ and p53^–/– HCT116 cells to LY. Cells were treated with LY or, as a control, with vehicle (DMSO), and cell numbers were assessed. Measurements were performed in triplicates. (c) Analysis of HCT116-derived knockout cell lines. Shown are the results of Western blot analysis of p53 and p21 protein levels after addition of adriamycin at 0.2 μg/ml for 8 hours. (d) p21 promoter reporter activity in p53-deficient HCT116 cells after treatment with 1.5 μM LY for 12 hours. The experiment was performed in triplicates. For details, see the Methods. (e) p53-independent induction of p21 mRNA by LY. Shown are the results of Northern blot analysis with RNA isolated at the indicated time points after addition of 1.5 μM LY to p53-deficient HCT116 cells. (f) p53-independent induction of p21 protein levels by LY. Shown are the results of Western blot analysis with lysates from p53^–/– HCT116 cells treated with 1.5 μM LY. Membranes were probed with antibodies specific for p21 and, as a loading control, α-tubulin. For details, see the Methods.