Figure 4.

Tivantinib blocks NLRP3 inflammasome assembly by weakening NLRP3 ATPase activity

(A) Qualification of intracellular potassium level by ICP-OES in LPS-primed BMDMs treated with various concentrations of Tivantinib (as labeled) and then stimulated with 5 μM nigericin for 30 min.

(B) Confocal microscopy analysis in LPS-primed BMDMs treated with 5 μM Tivantinib and then stimulated with 5 μM nigericin, followed by staining with Mitosox and DAPI.

(C) The relative fluorescence intensity of mitoSOX in LPS-primed BMDMs treated with 5 μM Tivantinib and then stimulated with 5 μM nigericin.

(D) Western blot analysis of ASC oligomerization in cross-linked cytosolic pellets and input of BMDMs treated with 5 μM Tivantinib and then stimulated with various concentrations of nigericin (as labeled) for 30 min.

(E) Immunoprecipitation (IP) and western blot analysis of the interaction of endogenous ASC and NLRP3 in LPS-primed BMDMs treated with 5 μM Tivantinib and then stimulated with 5 μM nigericin for 30 min.

(F and G) Western blot analysis of NLRP3 oligomerization by SDD-AGE assay in BMDMs treated with various concentrations of Tivantinib (as labeled) and then stimulated with 5 μM nigericin for 30 min. (G) IP and western blot analysis of the interaction between Flag-NLRP3 and VSV-NLRP3 in the lysates of HEK-293T cells treated with 10 μM Tivantinib.

(H) ATPase activity assay for purified NLRP3 in the presence of different concentrations of Tivantinib (as labeled).

(I) Docking complex of NLRP3 with Tivantinib. Tivantinib is shown in sticks and colored light green, NLRP3 is shown in cartoon and colored light gray, key amino acid residues were shown as sticks.

(J) MST assay for the affinity between Tivantinib and purified NLRP3 protein. Data represent means ± SEM from four biological duplicates (A, C, H). Statistical analysis was performed using one-way ANOVA(A, H) or unpaired Student’s t test (C). ∗p < 0.05, ∗∗∗p < 0.001.