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. 2020 Sep 30;11:565924. doi: 10.3389/fimmu.2020.565924

Figure 4.

Figure 4

Unprimed NLRP3 inflammasome activation has the same requirements as the canonical inflammasome. (A) WT and NLRP3 KO THP-1 cells were left unprimed prior to treatment with nigericin (10 µM, 2 h) to activate NLRP3 inflammasome. (B) Primary human monocytes were pre-incubated with MCC950 (1 µM) for 15 min prior to treatment with nigericin (10 µM, 45 min). The NP-40 soluble fraction and DSS crosslinked insoluble fraction were immunoblotted for ASC monomers, dimers and oligomers. Blots are representative of 3 independent biological experiments and blood donors. (C, D) THP-1 cells were left unprimed and pre-incubated with high K+ buffer, NPPB (50 µM) or NBC19 (25 µM) for 15 min prior to treatment with nigericin (10 µM, 45 min). (E, F) THP-1 cells were left unprimed pre-incubated with P22077 (2.5 µM, 15 min) prior to treatment with nigericin (10 µM, 45 min). n=3 independent biological replicates, mean ±; S.D., ***P < 0.001; ****P < 0.0001; ns (not significant) using one-way ANOVA comparing each sample to nigericin only treated sample. (D, F) Supernatants and lysates were analyzed for mIL-18 (18 kDa), pro-IL-18 (24 kDa), mCaspase-1 (20 kDa), pro-Caspase-1 (45 kDa), as well as loading control β-actin (42 kDa). Blots are representative of at least 3 independent experiments and in the case of primary monocytes, blood donors.