Knockdown of Puma by shRNA prevents sensitization to FAS- and Dex-induced apoptosis. A, CEM-6E2/p16 cells were infected with pMSCV-shPuma or the empty control vector (shCtr). Single cell clones were isolated by limiting dilution from bulk-selected CEM/p16-shPuma cells. To determine the knockdown of endogenous Puma, CEM/p16-shPuma cells were cultured in the absence or presence of 250 ng/ml Dox for 24 h, and lysates were analyzed by immunoblot. Two clones that induced p16INK4A but not endogenous Puma were selected. Equal protein loading was confirmed by α-tubulin staining. B, mitochondrial (mito) and cytoplasmic (cyto) extracts were prepared from CEM/p16-shCtr and CEM/p16-shPuma cells (clone 3) that were cultured in absence or presence of 250 ng/ml Dox for 24 h and treated with 10 nm Dex for another 40 h. The distribution of Puma, BAX, Cox4, and α-tubulin in subcellular fractions was detected by immunoblot. C and D, CEM/p16-shCtr and the shPuma-expressing clones CEM/p16-shPuma clone 3 and 6 were cultured with 250 ng/ml Dox for 24 h and then treated with 0.1 μg/ml anti-FAS antibody for 4 h or with 10 nm Dex for 24 h, respectively. Apoptosis induction was measured by PI-FACS analysis. Shown is the mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 for CEM/p16-shPuma clones treated either with +CH11 + Dox or +Dex + Dox compared with identically treated CEM/p16-shCtr control cells.