Fig 2: The NLRP1 CARD forms an ASC-containing inflammasome.(A) HEK 293TCASP1 + GSDMD cells were transfected with plasmids encoding the UPA-CARD fragments of NLRP1 or CARD8 and ASC as indicated. After 24 h, lysates were evaluated by immunoblotting. (B, C) HEK 293TCASP1 + GSDMD were transfected with plasmids encoding full-length NLRP1 or NLRP1 without a pyrin domain (NLRP1?PYD). The next day, the cells were pretreated with DMSO or bortezomib (bort., 20 µM, 30 min), which inhibits inflammasome formation, before the DMSO or VbP (10 µM, 6 h). (B, C) Supernatants were evaluated for LDH release (B) and lysates were analyzed by immunoblotting (C). Data are means ± SEM of three biological replicates. **P < 0.01, ***P < 0.001 by two-sided t test. (D, E) HEK 293T cells were transfected with plasmids encoding GFP-tagged ASC and the UPA-CARD fragments of NLRP1 or CARD8, and then evaluated for ASC speck formation by fluorescence microscopy. The cells were not fixed before analysis. (D, E) Shown are the mean ± SEM (D) and representative images (E) from 10 replicates from one of two independent experiments. ***P < 0.001, by two-sided t test. (F) Cell lysates from HEK 293T cells transiently expressing SmBiT-tagged full-length ASC were mixed with lysates expressing the indicated LgBiT-tagged CARD and analyzed for the relative luminescence. All data, including immunoblots, are representative of three or more independent experiments. NS, not significant.

Fig 3: NLRP1 is ASC-dependent and CARD8 is ASC-independent.(A) Human NLRP1, CARD8, and ASC domain organization. The autoproteolysis sites are indicated. (B, C) HEK 293T cells stably expressing CASP1 and GSDMD (HEK 293TCASP1 + GSDMD) were transiently transfected with constructs encoding the indicated proteins and treated with DMSO or VbP (10 µM, 6 h). (B, C) Supernatants were evaluated for LDH release (B) and lysates were analyzed by immunoblotting (C). Data are means ± SEM of three biological replicates. ***P < 0.001 by two-sided t test. (D, E) HEK 293T cells were transfected with constructs encoding GFP-tagged ASC and NLRP1 or CARD8, treated with DMSO or VbP (10 µM, 6 h), and evaluated for ASC speck formation by fluorescence microscopy. The cells were not fixed before analysis. (D, E) Shown are the mean ± SEM (D) and representative images (E) from 10 replicates from one of two independent experiments. ***P < 0.001 by two-sided t test. (F) HEK 293T cells transiently transfected with constructs encoding the indicated proteins and treated with DMSO or VbP (10 µM, 6 h). Lysates were harvested, subjected to disuccinimidyl suberate cross-linking, and evaluated by immunoblotting. All data, including immunoblots, are representative of three or more independent experiments. FL, full-length; WCL, whole cell lysate.

Fig 4: DPP8/9 inhibitors induce pyroptosis in primary lymphocytes.a Domain structures of human NLRP1 and CARD8. Note that the ZU5 and UPA sub-domains together are referred to as a FIIND. b, c The indicated primary cells were treated with VbP (24 h) before cell death was assessed by Cell-TiterGlo (b) and lysates were evaluated by immunoblotting (c). Data are means ± s.e.m. of four biological replicates. d CD3+ T cells were treated with VbP (20 µM, 24 h) or 8j (20 µM, 24 h) before lysates were evaluated by immunoblotting. Immunoblots are representative of three independent experiments.

Fig 5: LRRFIP1 and FLII negatively regulate human NLRP1 inflammasome activation A HEK293T cells were transfected with 300 ng NLRP1‐FLAG, 1 ng ASC‐GFP and the indicated concentrations of FLAG‐LRRFIP1 and FLAG‐FLII plasmids, and the formation of ASC specks was analyzed by fluorescence microscopy 24 h post‐transfection.B, C Representative images of ASC specks.D, E Number of positive ASC specks in cells co‐transfected with LRRFIP1 (D) and FLII (E). Expression of LRRFIP and FLII was confirmed by Western blot using anti‐FLAG and anti‐ACTB antibodies. Data are shown as the means ± SEM (n = 95–156 cells). P values were calculated using one‐way ANOVA and Tukey's multiple range test. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure.