FIG. 3.
RIP is required for TNF-induced apoptosis but not for Fas-L- or TRAIL-induced apoptosis. (A) HeLa control, TRADD KD, or RIP KD cells were preincubated with cycloheximide (10 μg/ml) and then treated with TNF (20 ng/ml), CH-11 antibody (250 ng/ml), or TRAIL (10 ng/ml) for 4 h, respectively. Apoptosis of HeLa control, TRADD KD, or RIP KD cells was determined by PI staining and flow cytometry. (B) HeLa control, TRADD KD, or RIP KD cells were preincubated with cycloheximide (10 μg/ml) and then treated with TNF (20 ng/ml), CH-11 antibody (250 ng/ml), or TRAIL (10 ng/ml) for 4 h, respectively. Apoptosis of HeLa control, TRADD KD, or RIP KD cells was determined by the active caspase 3 assay. (C) HeLa control, TRADD KD, or RIP KD cells were preincubated with cycloheximide (10 μg/ml) and then treated with TNF (20 ng/ml) for 3, 6, or 16 h (T3, T6, and T16, respectively). Apoptosis was determined by the active caspase 3 assay. (D) HeLa control, TRADD KD, or RIP KD cells were preincubated with cycloheximide (10 μg/ml) and then treated with TNF at 2, 10, or 50 ng/ml for 6 h (T2, T10, and T50, respectively). Apoptosis was determined by the active caspase 3 assay. (E) pcDNA3.1 control vector or pcDNA3.1-m-IκB was cotransfected with EGFP at a 10:1 ratio into HeLa control, TRADD KD, FADD KD, or RIP KD cells. After 48 h, cells were treated with either TNF (20 ng/ml) or CH-11 (100 ng/ml) for 4 h. Apoptosis was then analyzed by the active caspase 3 assay. The percentage of apoptotic cells from three independent determinations with the corresponding standard deviation is indicated. (F) HeLa control, TRADD KD, FADD KD, or RIP KD were preincubated with cycloheximide (10 μg/ml) and then treated with TNF (20 ng/ml) for 4 h. Whole-cell extracts were then prepared and analyzed by Western blotting with PARP antibody. (G) HeLa cells were preincubated with z-VAD-fmk (20 nM) and then treated with TNF (10 ng/ml), CH-11 antibody (100 ng/ml), or TRAIL (10 ng/ml) plus cycloheximide (10 μg/ml) for 4 h. Apoptosis was then analyzed by PI staining and flow cytometry. The percentage of apoptotic cells from three independent determinations with the corresponding standard deviation is indicated.