Fig. 7. BEL treatment induces p53-dependent apoptosis in p21-deficient HCT cells. (A) HCTp21^-/- cells have a high death rate after inactivation of iPLA₂ by BEL. HCTp21^-/- cells (5x10⁵) were treated with varying concentrations of BEL for 28 hours. Viable and nonviable cells were determined by Trypan Blue staining and counted. Error bars represent the mean ± s.d. of six experiments. (B) Inactivation of iPLA₂ induces apoptosis of p21-deficient cells. Both wild-type HCT and HCTp21^-/- cells were incubated with or without BEL for 10 hours and then analyzed for apoptosis by Annexin-V-Fluos staining and FACS. R1 represents living cells. R2 represents cells undergoing early apoptosis, and R3 representes secondary necrotic cells. Each panel shows a typical flow-cytometric histogram of 10,000 cells/sample from one of three experiments. (C) BEL-associated death of HCTp21^-/- cells can be prevented by p53 depletion. HCTp21^-/- cells were cultured overnight in the presence of BEL with 1 nmol of SMARTpool-p53. dsRNA was used as a negative control. The cells were harvested for both western blot analysis for p53 (top) and cell death analysis (bottom). Recombinant TNT p53 was loaded as indicated. ^*P<0.05.