A. E2F-1 expression up-regulates the activity of the PUMA promoter in HCT116 PUMA+/+ cells. HCT116 PUMA+/+ cells were co-transfected with a total of 0.82 μg of DNA, including 0.8 μg of PUMA promoter reporter plasmid and 0.02 μg phRL-CMV plasmid (as an internal control) in a 12-well plate by using Lipofectamine 2000 transfection reagent, and then processed as in Fig. 2A. 4B Up-regulation of PUMA at the protein level after Ad-E2F-1 expression in HCT116 PUMA+/+ cells. Western blot analysis of PUMA was performed in HCT116 PUMA+/+ cells at indicated time points as in Fig. 1B. PUMA/actin showed the ratio of the scanned optical density of each protein band. PUMA protein level was increased at 12 hours and 16 hours of infection with Ad-E2F-1.