Figure 4.

(See previous page) . p53-independent induction of PUMA in response to cytotoxic effect of H1. HCT116 and HCT116 p53^−/− cells were treated with various concentrations of H1 for 24 h, and cells were harvested. Induction of PUMA by H1 was determined by western blot analysis (A and B). Effect of H1 on the expression of Bax and Bcl-2 also detected (A and C). In addition, cells were exposed to H1 for different time points (0, 4, 8, 12, 24 h), after that PUMA expression was determined by western blot analysis (E and F). siRNA specific targeting to PUMA was mixed with Lipofectamine 2000 (LF2000, Invitrogen) in serum-free RPMI-1640 medium and added into plated cells. After 24 h incubation, cells were treated with H1 for additional 24 h. Then, cells were rinsed and lysed containing freshly added Protease Inhibitor Cocktail. Cell lysates were used for western blot analysis (F). HCT116 p53^−/− cells were seeded into 96-well plate at density of 1000 cells per well, then transfected with 10nM siPUMA mixed with LF2000 added into the well. After 24 h transfection, H1 (4μM) was added and exposed for an additional 72 h, and then cell viability was determined by MTT assay (G). **P < 0.01; ^##P < 0.01 vs. each control group.