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. Author manuscript; available in PMC: 2020 Nov 3.
Published in final edited form as: Nat Chem Biol. 2019 May 13;15(6):560–564. doi: 10.1038/s41589-019-0278-6

Figure 1. MCC950 closes the conformation of autoinflammatory-associated gain-of-function NLRP3.

Figure 1

a,b, BRET signal in HEK293T cells expressing wild-type or p.D305N YFP-NLRP3-Luc as indicated, incubated for 24 h with different concentrations of MCC950 (a) or for different times with MCC950 (10 μM) (b). Vehicle control data is concentration zero (a) and time zero (b). Centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility. c, BRET signal (bottom) and immunoblots for NLRP3, IL-1β, GSDMD and β-actin (top) from immortalized Nlrp3 -/- macrophages expressing YFP-NLRP3-Luc p.T350M or p.D305N, treated for 16 h with doxycycline (1 μg/ml) and LPS (100 ng/ml) alone or in combination with MCC950 (10 μM). Blots are representative of n=2 independent experiments with similar result (full blots are shown in Supplementary Figure 10a) and for BRET assays centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility; Mann-Whitney test, two-tailed, ***p<0.0001 (U=0). d, Quantification of the number of mutant NLRP3-YFP oligomers per HEK293T cell incubated or not for 24 h with MCC950 (10 μM). Data are representative of n=3 independent experiments and centre values represent mean and error bars the SEM from 1000-1500 cells. e, Percentage of ASC-specking monocytes identified by time of flight assay (left) and ELISA for IL-1β release (right) from PBMCs isolated from healthy donors (white) and individuals with CAPS carrying the NLRP3 p.D305N (grey) after 4h incubation with LPS (1 μg/ml). Centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility. f,g, Inhibition of the percentage of ASC-specking monocytes (f) and IL-1β release (g) from PBMCs after 4h incubation with LPS (1 μg/ml) alone (for CAPS samples) or followed by nigericin (5 μM, 30 min, for healthy individuals) in the presence of different concentrations of MCC950. Centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility.