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

Figure 3.

Figure 3

mIL-18 release in the absence of priming is NLRP3 dependent. THP-1 cells (A, B) and monocytes (C, D) were pre-incubated with MCC950 (1 µM) or Z-VAD (50 µM) for 15 min prior to treatment with nigericin (10 µM, 45 min). (A, C) Secreted IL-18 was measured by ELISA and cell death was measured by LDH assay and shown as percentage relative to total cell death, n=3 independent biological replicates for THP-1 and n=6 independent blood donors for human monocytes, mean ± S.D., *P < 0.05; **P < 0.01; ns (not significant) using one-way ANOVA comparing each sample to the nigericin only treated sample. (E, F) Unprimed WT or NLRP3 KO THP-1 were treated with nigericin (10 µM, 45 min). (E) Secreted IL-18 was measured by ELISA and cell death was measured by LDH assay and shown as percentage relative to total cell death, n=3 independent biological replicates, mean ± S.D., * = P < 0.05; ns (not significant) using two-way ANOVA comparing nigericin treated WT and NLRP3 KO THP-1s. (B, D, F) Western blot analysis for mIL-18 (18 kDa), pro-IL-18 (24 kDa), mCaspase-1 (20 kDa), pro-Caspase-1 (45 kDa), and in some cases NLRP3 (113 kDa), as well as loading control β-actin (42 kDa). Blots are representative of at least 3 independent biological experiments and blood donors.