Figure 4

Ceramide mediates TRAIL/zVAD/CHX- and TNF/zVAD/CHX-induced programmed necrosis in the examined sensitive tumor cell lines. ( a ) Cells were left untreated or stimulated with TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b for the indicated times before intracellular ceramide levels were determined in duplicate. Raw data from the charred TLC plates (C16 and C18 ceramide) are shown below the bar graphs. Loss of membrane integrity as a marker for programmed necrosis was determined in parallel by trypan blue staining and is shown above the respective bars. ( b ) Cells were left untreated or preincubated with 10 μM Arc39 for 2 h before addition of TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b. After 24 h of stimulation, programmed necrosis was analyzed by flow cytometric analysis of PI-positive cells. ( c ) Wild-type (WT) and RIPK3-deficient (RIPK3^−/−) primary MEF were left untreated or preincubated with 10 μM Arc39 for 2 h with subsequent addition or not of 100 ng/ml of TRAIL or TNF in combination with 20 μM zVAD-fmk and 1 μg/ml CHX. After 16 h, programmed necrosis was analyzed by flow cytometric analysis of PI-positive cells.