Figure 3.

Endogenous Itaconate Regulates NLRP3 Activation and 4-OI Dicarboxypropylates C548 on NLRP3

(A and B) LPS (3 h) and nigericin (45 min) induced IL-1β release (A, n = 6) and LDH release (B) in wild-type (WT, n = 7) and Irg1^−/− (n = 8) BMDMs.

(C and D) Immunoblot analysis (C) and quantification by densitometry (D, n = 4) of pro- and mature caspase-1 and IL-1β protein in lysates and supernatants of wild-type (WT) and Irg1^−/− BMDMs treated with LPS (3 h) and nigericin (45 min).

(E) IL-1β release from NLRP3 inflammasome-reconstituted HEK293T cells treated with nigericin (45 min) (n = 3). Immunoblot analysis of pro- and mature IL-1β and IRG1 protein in supernatants and lysates of these cells.

(F) Endogenous co-immunoprecipitation of NLRP3 and NEK7 in BMDMs treated with LPS (3 h) and nigericin (45 min) ± 4-OI (250 μM).

(G) Tandem mass spectrometry spectrum of C548-containing NLRP3 peptide following 4-OI treatment (250 μM, 24 h).

^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Data are mean ± SEM. Blots are representative of a minimum of 3 independent experiments.