Figure 4. RIPK3 kinase activity is not required for MLKL-independent activation of NLRP3.

(a) WT littermate and Caspase-8^LysMcre BMDM were primed for 3 h with Pam₃Cys (2 μg ml⁻¹), and as indicated RIPK3 inhibitor (R3 inhib GSK872; 1 μM) was added in the last 20 min of priming. Cells were then exposed to Cp.A (500 nM), as specified, for a further 24 h. Levels of IL-1β secretion were measured by ELISA. n=4 mice per genotype, mean+s.e.m. (b,c) WT, Ripk3^−/− and Mlkl^−/− BMDM were primed for 3 h with LPS (20 ng ml⁻¹) in the absence or presence of RIPK3 inhibitor (R3 inhib; 1 μM), prior to addition of Cp.A for a further 6 h. Supernatants were assayed for (b) IL-1β and (c) TNF by ELISA or death assessed by lactate dehydrogenase (LDH) activity (see Supplementary Fig. 2f). n=3 mice per genotype, mean+s.e.m. (d,e) Cell supernatants (d) and lysates (e) from WT, Mlkl^−/− and Ripk3^−/− BMDM primed with LPS (3 h) and treated with Q-VD-OPh (20 μM) and R3 inhibitor (1 μM, last 20 min of priming), as indicated, and subsequently treated with Cp.A (1 μM, 5 h) were analyzed by immunoblot as indicated. One of three experiments. Full-size immunoblots are presented in Supplementary Fig. 12. (f) WT and caspase-1^−/− BMDM were primed for 3 h with LPS, and as indicated treated with Q-VD-OPh (20 μM) and R3 inhib (1 μM) for the last 20 min of priming. BMDM were then cultured with Cp.A (1 μM) or Alum (300 μg ml⁻¹) for a further 6 h. Culture supernatants were assayed for IL-1β levels by ELISA. n=3 mice, mean+s.e.m., one of two experiments. (g–i) WT, Ripk3^−/− and Mlkl^−/− BMDM were primed for 3 h with LPS (20 ng ml⁻¹) in the presence of Q-VD-OPh (20 μM), and where indicated 1 μM RIPK3 inhibitor (R3 inhib), prior to addition of Cp.A for a further 6 h. Supernatants were assayed for (g) IL-1β and (i) TNF by ELISA, and (h) cell death was measured via an LDH assay. n=3 mice per genotype, mean+s.e.m. *NS, non-specific band.