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

Figure 2.

Figure 2

TAK1 and NF-κB differentially contribute to unprimed inflammasome activation in human monocytes. (A, B) Primary human monocytes were pre-incubated with 5z-7-Oxozeaenol (0.5 µM) for 15 min prior to treatment with nigericin (10 µM, 45 min). (A) 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=5 independent biological replicates (each point represents a different blood donor). Error bars represent mean ± S.D., *P < 0.05 using one-way ANOVA comparing each sample to the nigericin only treated sample. (B) 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. (C) Primary human monocytes were left untreated or pre-treated with JSH-23 (40 µM, 13 h) or SB220025 (20 µM, 30 min) followed by treatment with nigericin (10 µM, 45 min). Cell supernatants were assayed for the release of IL-18, cell death by LDH release and caspase-1 activity by caspase-1-Glo assay. n=4 independent biological replicates (each point represents a different blood donor). Error bars represent 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. (D, E) Primary human monocytes were left untreated or pre-treated with JSH-23 (40 µM, 13 h) or SB220025 (20 µM, 30 min) following treatment with LPS (1 µg/ml, 4 h) or left untreated. (D) Cell supernatants were assayed for TNF-α and IL-6 release. n=5 independent biological replicates (each point represents a different blood donor). Error bars represent mean ± S.D., *P < 0.05 using one-way ANOVA comparing each sample to the LPS only treated sample. (E) Lysates were analyzed for proIL-18 (24 kDa) and NLRP3 (113 kDa) as well as loading control β-actin (42 kDa). (F) Western blot analysis of human monocyte lysates for IκBα (39 kDa) and loading control β-actin (42 kDa) from cells left untreated or treated with nigericin (10 µM, 45 min). All blots are representative of 2 independent biological experiments (each from a different blood donor).