Fig. 1 ∣. Esterified Xaa-Pro dipeptides selectively activate the CARD8 inflammasome.
a, Domain organizations of human NLRP1 and CARD8. Both proteins undergo autoproteolysis between the ZU5 and UPA subdomains of the FIIND. b, Inhibition of recombinant DPP9 activity by the indicated compounds. IC50 values are shown in parentheses. c, The indicated MV4;11 cells were treated with compounds (1 mM) and monitored for PI uptake. Ft, Fluorescence measurement at time t; F0, fluorescence measurement at time 0; Fmax, maximum fluorescence measurement obtained in the experiment. d,e, MV4;11 (d) or THP-1 (e) cells were treated with XP-OMes (1 mM) or VbP (2 μM) for 14 h (d) or 24 h (e) before LDH release and immunoblot analyses. f, MV4;11 cells were treated with VP-OMe (2 mM), IP-OMe (1 mM), bortezomib (bort; 1 μM) and/or VX-765 (50 μM) as indicated for 6 h before LDH release and immunoblot analyses. ***P < 0.001, **P < 0.01 by two-sided Students t-test. NS, not significant. The indicated significant P values are as follows: VP-OMe versus DMSO = 0.0054, IP-OMe versus DMSO = 0.0020. g, N/TERT-1 immortalized keratinocytes were treated with XP-OMes (1 mM) or VbP (10 μM) for 16 h before LDH release and immunoblot analyses. h, HEK 293T cells were transfected with plasmids expressing FLAG-tagged NLRP1 and GFP-tagged ASC and treated with XP-OMes (1 mM) or VbP (10 μM) for 24 h. ASC speck formation was assessed by fluorescence microscopy. Representative images and average cells with specks (%) ± s.e.m. are shown. Scale bar, 100 μm. h, RAW264;7 cells were treated with XP-OMes (1 mM) or VbP (2 μM) for 24 h before LDH release and immunoblot analyses. Data in b (n = 4), c (n = 4) and d-i (n = 3) are presented as means ± s.e.m. of replicates. All data, including immunoblots, are representative of three or more independent experiments.