A. WT HCT116 cells were treated with 0.5 μM UCN-01, 50 μM cisplatin, or their combination for 24 hr. PUMA, p53, and active caspase 3 were analyzed by Western blotting. B. WT and PUMA-KO HCT116 cells were treated with 0.5 μM UCN-01, 50μM cisplatin, or their combination for 48 hr. Apoptosis was determined by nuclear staining with Hoechst 33258. C. p21-KO HCT116 cells were treated with 0.5 μM UCN-01, alone or in combination with an adenovirus (10 MOI) expressing WT PUMA (Ad-PUMA) or mutant PUMA with BH3 domain deletion (Ad-ΔBH3). Apoptosis was determined by nuclear staining 48 hr after treatment. D. p21-KO HCT116 cells were treated with 2μM UCN-01, alone or in combination with 1μM of the BH3 mimetic GX15-070. Apoptosis was determined by nuclear staining 48 hr after treatment. The results were averages of three independent experiments ± standard deviation.