Induction of programmed necrosis by TRAIL/zVAD/CHX and TNF/zVAD/CHX in human tumor cell lines. Cells were treated with 100 ng/ml of (
) TRAIL or (
) TNF in combination with 50 μM zVAD-fmk and non-toxic concentrations of CHX (U-937 0.1 μg/ml; Mz-ChA-1 2 μg/ml; BxPC-3 1 μg/ml; HT-29 5 μg/ml; Colo357 5 μg/ml; Panc89 1 μg/ml; PancTu-I 10 μg/ml; A818-4 10 μg/ml; CCRF-CEM 0.0625 μg/ml; MKN-28 5 μg/ml; SK-OV-3 1 μg/ml; KNS-62 5 μg/ml; Pt45P1 0.1 μg/ml; SK-MEL-28 10 μg/ml). After 24 h, loss of membrane integrity was measured as a marker for programmed necrosis by flow cytometric detection of PI-positive cells. Values above the respective columns represent the specific percentage of programmed necrosis (stimulus-induced minus zVAD/CHX-induced programmed necrosis), spontaneous cell death in untreated cells is shown for comparison. (
) Cells were treated with their respective LD50 concentrations of CHX alone (CCRF-CEM 60 μg/ml; MKN-28 1000 μg/ml; SK-OV-3 500 μg/ml; KNS-62 300 μg/ml; Pt45P1 150 μg/ml; SK-MEL-28 625 μg/ml) or in combination with 100 ng/ml TRAIL or TNF and 50 μM zVAD-fmk. After 24 h, viability was determined by crystal violet staining (for the adherent cell lines MKN-28, SK-OV-3, KNS-62, Pt45P1 and SK-MEL-28) or XTT assay analysis (for the suspension cell line CCRF-CEM). (
) Cells were left untreated or stimulated with TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b in the presence of the indicated concentrations of the Smac mimetic birinapant. After 8 or 24 h of stimulation, programmed necrosis was analyzed by flow cytometric analysis of PI-positive cells.