Figure 5. Deletion of both RIPK3 and caspase-8 abrogates TLR- and Cp.A-induced activation of caspase-1 and IL-1β.

(a) WT, Mlkl^−/−, Ripk3^−/− and Ripk3^−/−Caspase-8^−/− BMDM were primed with Pam₃Cys for 3 h and incubated with Alum (300 μg ml⁻¹) for 6 h, and ATP (5 mM) or nigericin (10 μM) for 40 min. Supernatants were assayed for IL-1β release. n=3 mice per genotype, mean+s.e.m., one of three experiments. (b) WT, Ripk3^−/− and Ripk3^−/−Caspase-8^−/− BMDM were primed with Pam₃Cys for 3 h and cultured with Nigericin as indicated for 40 min. Supernatants and lysates were analyzed by immunoblot. One of two experiments. Full-size immunoblots are presented in Supplementary Fig. 13. (c) WT, Mlkl^−/−, Ripk3^−/− and Ripk3^−/−Caspase-8^−/− BMDM were primed with Pam₃Cys for 3 h and incubated with increasing concentrations of Cp.A for 24 h and supernatants and lysates were analyzed by immunoblot. One of two experiments. Full-size immunoblots are presented in Supplementary Fig. 13. (d) WT, Mlkl^−/−, Ripk3^−/− and Ripk3^−/−Caspase-8^−/− BMDM were primed with Pam₃Cys for 3 h, and as indicated Q-VD-OPh (20 μM) for the last 20 min of priming, and then cells were treated where shown with Cp.A for a further 6 h. Supernatants were assayed for IL-1β levels. n=3 mice per genotype; mean+s.e.m., representative of one of three experiments. (e) Unprimed WT and Ripk3^−/−Caspase-8^−/− BMDM were cultured with Nigericin (10 μM, 2 h), Cp.A (1 μM, 20 h) and ATP (5 mM, 2 h), and supernatants and lysates were analyzed by immunoblot for caspase-1 activation and NLRP3 levels. n=3 mice per genotype (numbered). Full-size immunoblots are presented in Supplementary Fig. 13. (f) Unprimed WT, Ripk3^−/−, Ripk3^−/−Caspase-8^−/− and Nlrp3^−/− BMDM were stimulated with Nigericin (10 μM, 2 h), Cp.A (1 μM), and ATP (5 mM, 2 h) as indicated, and supernatants and cell lysates were analyzed by immunoblot. One of three experiments. Full-size immunoblots are presented in Supplementary Fig. 13. (g) WT BMDM were primed with LPS (20 ng ml⁻¹) for 3 h, stimulated with Nigericin or ATP, and lysates analyzed by immunoblot. (h) Schematic depicting how RIPK3 signals NLRP3–caspase-1 and IL-1β activation based on the data presented in Figs 1, 2, 3, 4, 5. *NS, non-specific band.