Fig. 6: Mitochondrial complex I inhibition is necessary for CL097 activation of NLRP3 inflammasome.

a, Percent LDH release from BMDMs treated with LPS (100 ng ml^–1) and CL097 (70 μM), with or without VX-765 (20 μg ml–1); n = 4. b, OCR of BMDMs treated with LPS (100 ng ml^–1), with or without VX-765 (20 μg ml^–1). Data is shown as a percent of the basal OCR of untreated. CL097 (70 μM), Oligomycin (2 μM) and 2-deoxy-d-glucose (2DG) (50 mM) were added at indicated timepoints; n = 4. c,d, OCR of WT and NDI1 BMDMs treated with LPS (100 ng ml^–1). Data are shown as a percentage of the basal OCR of untreated. Piericidin A (500 nM) (c) or CL097 (70 μM) (d), Oligomycin (2 μM) and 2DG (50 mM) were added as indicated; n = 3 for each genotype. e, IL-1β protein levels in cell culture supernatant of WT or NDI1 BMDMs treated LPS (100 ng ml^–1) and CL097 (70 μM); n = 6. f, Intracellular caspase-1 (p20 fragment) protein expression in cell lysates from WT and NDI1 BMDMs treated as in Fig. e (70 μM); n = 6. g, Intracellular pro-caspase-1 expression in cell lysates treated as in e; n = 6. h, IL-1β protein levels in cell culture supernatant of WT BMDMs treated with LPS (100 ng ml^–1) and CL097 (70 μM), piericidin A (100 nM), myxothiazol (100 nM), antimycin A (100 nM) or oligomycin (50 nM); n = 4. i, IL-1β protein levels in cell culture supernatant of BMDMs treated with LPS (100 ng ml^–1) and CL097 (70 μM), with or without piericidin A (500 nM); n = 4. j, Intracellular pro-caspase-1 protein expression in cell lysates from BMDMs treated as in i; n = 4. k, Intracellular caspase-1 protein expression in cell lysates from BMDMs treated as in i; n = 4. l, IL-1β protein levels in cell culture supernatant of BMDMs treated with LPS (100 ng ml^–1) and CL097 (70 μM), with or without CyCr (10 μM) (n = 4). m, Intracellular caspase-1 protein expression in cell lysates treated as in l; n = 4. n, Intracellular pro-caspase-1 protein expression in cell lysates from BMDMs treated as in l; n = 4. Data are means ± s.e.m. *P < 0.05, two-tailed t-test (i, *P = 0.007; l, *P = 0.0127) one-way ANOVA with Tukey’s post hoc test for multiple comparisons (a, *P = 0.0399 UT/LPS + 20 min; *P < 0.0001 UT/LPS + 30 min, UT/LPS + 60 min, LPS + 30 min/LPS + 30 min+VX-765, LPS + 60 min/LPS + 60 min + VX-765; *P = 0.0013 (e); *P < 0.0001 (f); *P = 0.0273 (j); *P < 0.0001 (m)).

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