Fig 2: Specific CARD–CARD interactions determine ASC-dependent or independent inflammasome assembly.(A) Expression of the indicated LgBiT-tagged CARDs in HEK 293T cells was verified by immunoblotting. (B, C) Cell lysates from HEK 293T cells transiently expressing SmBiT-tagged ASCCARD (B) or SmBiT-tagged CASP1CARD (C) were mixed with lysates expressing LgBiT-tagged CARDs and analyzed for the relative luminescence. Data are means ± SEM of four independent replicates.

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: 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 5: Human pro-caspase-1 interdomain linker cleavage is required for pyroptosis.(A) HEK 293T cells stably expressing GSDMD were transiently transfected with plasmids encoding RFP (mock), the indicated pro-caspase-1 constructs, and the CARD8UPA-CARD for 24 h before lysates were analyzed by immunoblotting. Asterisks indicate background bands. (B) HEK 293T cells stably expressing the indicated pro-caspase-1 constructs were transiently transfected with plasmids encoding CARD8UPA-CARD or NLRP1UPA-CARD and ASC for 24 h before lysates were evaluated by immunoblotting. (C, D) Control and CASP1-/- THP-1 cells ectopically expressing the indicated pro-caspase-1 proteins were treated with DMSO or VbP (10 µM, 24 h). (C, D) GSDMD cleavage was assessed by immunoblotting (C) and cell death by LDH release (D). Data are means ± SEM of three biological replicates. ***P < 0.001 by two-sided t test. An asterisk indicates background bands. All data, including immunoblots, are representative of three or more independent experiments. CL, cleaved; FL, full-length.