Figure 4

Increased cell death following IAP knockdown due to apoptosis. Cells were transfected with the combination of all three siRNAs targeting cIAP-1 (0.5 nM), cIAP-2 (0.5 nM), and XIAP (0.5 nM) (siTarget), or corresponding non-targeting control siRNA (0.5 nM of each control siRNA to a final concentration of 1.5 nM) (siCon). 24 hour post-transfection cells were treated with TRAIL (25 ng/ml) for a further 24 hours. Cell death was assessed by Annexin V/PI DNA staining and flow cytometry. A. Representative histograms following combined knockdown of all three IAPs (siTarget) +/- TRAIL treatment. B. Percent of cells in each quadrant (R1: PI/AV +/-, Necrotic; R2: PI/AV +/+, Late apoptotic/Necrotic; R3: PI/AV -/-, Live cells; R4: PI/AV -/+, Early apoptotic cells). Representative of three independent experiments. C. Cells were pre-treated with zVAD.fmk (100 μM) for 1 hour prior to TRAIL treatment for 24 hours. Viability was assessed by PI-DNA staining and flow cytometry. D. Following TRAIL treatment for 9 hours, cell lysates were analysed for PARP processing by Western blot. Western shown is a representative of three independent western blots.