Fig. 6.

CASP8-RIPK1-RIPK3 axis regulates NLRP3 inflammasome. (A) Immunoblot of GSDMD with whole cell lysates in the indicated conditions at 6 h. (B, D) IL-1β protein concentrations in culture supernatants were measured by enzyme-linked immunosorbent assay after inhibition of CASP8, RIPK1, and/or RIPK3 (B) or after additionally adding ATP (5 mM) (D) (n = 4–6 healthy donors). (C) Immunoblot of NLRP3, IL-1β, and cleaved CASP1 using whole cell lysates in the indicated conditions at 6 h. The inhibitors targeting caspases, RIPK1, and RIPK3 are zVAD (50 μM), necrostatin-1 s (30 μM), and GSK872 (10 μM), respectively. (E) Immunoblot of cleaved CASP1 using whole cell lysates in the indicated conditions. (F) CXCL10, TNF, and IL12B mRNA was measured by qPCR and normalized relative to GAPDH mRNA (n = 5). Data are depicted as 1 of at least 3 representative blots (A, C, and E) and as mean ± SEM for the rest. ****P ≤ 0.0001; **P ≤ 0.01; *P ≤ 0.05 by 1-way analysis of variance with Tukey’s post hoc test for multiple comparisons (B, D, and F).