Figure 3

Role of RIPK1 and RIPK3 as determinants of susceptibility or resistance of tumor cell lines to programmed necrosis. ( a ) Cells were analyzed as in Figure 1a and b in the presence or absence of 50 μM necrostatin-1. ( b, c ) Western blots for expression of RIPK1 ( b ) and RIPK3 ( c ). Asterisks: non-specific bands. RIPK3 corresponds to the lower band of the doublet. A quantitative analysis of the relationship between the levels of RIPK3 and the specific sensitivity of the respective tumor cell line to TRAIL/zVAD/CHX-induced programmed necrosis (values taken from Figure 1a) is shown below. ( d ) U-937 and HT-29 cells were transfected with siRNAs specific for human RIPK3 (two individual siRNAs with distinct target sequences to rule out off-target effects) or a negative control siRNA (siCtr). 48 or 72 h after transfection, cells were left untreated or incubated with TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b. After 24 h, the decrease of intracellular ATP levels was analyzed as a marker for programmed necrosis. Insets: control Western blots of transfected, untreated cells for downregulation of RIPK3. ***P < 0.001. ( e ) Wild-type (WT) and RIPK3-deficient (RIPK3^−/−) primary MEF were stimulated with 100 ng/ml of TRAIL or TNF, 20 μM zVAD-fmk and 1 μg/ml CHX for 22 h before ATP levels were measured. Inset: Western blot for RIPK3. *P < 0.05, ***P < 0.001, n. s., not significant. ( f ) As part of the experiment shown in ( d ), U-937 and HT-29 cells were nucleofected with siRNA specific for MLKL. The data are shown in new panels, but with the same negative control (siCtr) values as in ( d ). Insets: control Western blots of transfected, untreated cells for downregulation of MLKL. ( g ) Cells were treated and analyzed as in Figure 1a and b in the presence or absence of 1 μM necrosulfonamide.