IKK2 expression induces the extrinsic apoptotic pathway. (A) Apoptosis detection in Ramos cells transfected with empty vector or CA-IKK2. Doxycycline (0.5 μg/mL) was added to the culture medium for 5 days before cells were analyzed for annexin-V binding. (B) Percentages of dead cells after induction of transgene expression without (▭) or in the presence of caspase inhibitor zVAD (▭). Viable and dead cell numbers were determined by trypan blue exclusion. Cells were treated with doxycycline and in the presence or absence of zVAD (20 μM) for 4 days. (C) Expression of CFLAR in Ramos-pRTS-GFP and Ramos-pRTS-CA-IKK2. Immunoblot analysis of Ramos cells transfected with pRTS-GFP or pRTS-CA-IKK2 and treated with doxycycline (0.5 μg/m) for 48 hours. Cell extracts were analyzed for CFLAR and as a control for actin expression. (D) Percentages of dead cells after transfection of Ramos-pRTS-GFP and Ramos-pRTS-CA-IKK2 with either pcDNA (control, ▭) or a CFLAR expression vector (▬). Cells were treated with doxycycline for 7 days to induce the pRTS-system. (E) Immunoblot analysis of Ramos-pRTS-CA-IKK2 cells stably transduced with shRNA expression vectors encoding either a scrambled shRNA (sh-co) or shRNA against caspase 8 (sh-C8). (F) Percentages of dead Ramos cells expressing the nonsense shRNA (▭) or the shRNA against caspase-8 (▬) after CA-IKK2 transgene expression. Cells were treated with doxycycline (0.5 μg/m) for 6 days. All error bars represent SDs from triplicate experiments.