Activation of caspase-9 was accompanied by PUMA-mediated E2F-1-induced apoptosis. 2 × 106 of SK-MEL-2 (A), HCT116 PUMA+/+ cells (B), and HCT116PUMA-/- cells (C) were infected with Ad-E2F-1 or Ad-LacZ and lysed at indicated time points to collect their intracellular contents. The cell lysates were assayed for caspase-9 activity by measuring the digestion of p-nitroanaline (pNA) as a substrate. The OD405 values were measured, and the percentage values were compared with Ad-LacZ infected cells as indicated. Caspase-9 activity was induced 3.6 fold, 3.0 fold, and 1.5 fold after 48 hours of Ad-E2F-1 infection in SK-MEL-2 cells, HCT116 PUMA+/+ cells, and HCT116PUMA-/- cells, respectively.