Figure 5.

RIPK3 promotes TNFα-induced INS-1 cell death when caspases are inhibited. A) NIT-1 CTL and NIT-1 RIPK3Δ cell death was quantified 48 h post treatment (n = 3). Cell culture treatment conditions are indicated by color (black: vehicle, blue: 40 ng/ml TNFα, green: 40 ng/ml TNFα + 50 μM zVAD) and β-cell lines are indicated by shapes (NIT-1 CTL: circles, NIT-1 RIPK3Δ: diamonds, INS-1: squares, INS-1 Empty: triangles, INS-1 mRIPK3: hexagons). B) NIT-1 CTL and RIPK3Δ cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 4). C) INS-1 cell death was quantified 24 h post treatment relative to t = 0 (n = 4–9). Cell culture treatment conditions are indicated by color (black: vehicle, white: 5 μM GSK’872, blue: 40 ng/ml TNFα, light blue: TNFα+GSK’872, green: TNFα + 50 μM zVAD, light green: TNFα+zVAD + GSK’872). D) INS-1 cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated cells (n = 3). E)Ripk3 RNA expression (n = 4) and F) RIPK3 protein expression were quantified in control (pcDNA3-Empty: triangles) and Ripk3 overexpressing (pcDNA3-mRipk3: hexagons) INS-1 cells. G) INS-1 pcDNA3-Empty and pcDNA3-mRipk3 cell death was quantified 24 h post TNFα or TNFα+zVAD treatment (n = 4). H) Immunoblot analysis of proteins immunoprecipitated with anti-RIPK3 following 24 h TNFα+zVAD treatment (n = 3). I) Immunoblot analysis of proteins immunoprecipitated with anti-MLKL following 24 h TNFα+zVAD treatment (n = 3). Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, p > 0.05 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)