Fig 2: Gasdermin D deletion prevents cell death in the liver of ACLF mice. (A) Hepatic protein expression of CASP3 in WT or GSDM-D KO mice with ACLF. (B) Hepatic protein expression of CASP4 in WT or GSDM-D KO mice with ACLF. (C) Hepatic protein expression of CASP11 in WT or GSDM-D KO mice with ACLF. (D) Hepatic protein expression of CASP1 in WT or GSDM-D KO mice with ACLF. (E) IL-1β (ELISA) and IL-18 (ELISA) levels in WT or GSDM-D KO mice with ACLF. (F) Representative images and quantification of RIPK3 immunofluorescence in the liver were measured as a percentage of positive area per field. Scale bar for 400×: 20 µm. Western blot data are represented as normalised to GAPDH levels and as the ratio between the cleaved form and the full-length form. Data are expressed as mean±SD (n=6–9 per group). Statistical significance was determined using two-tailed t-test: *p<0.05, **p<0.001. ACLF, acute-on-chronic liver failure; CASP, caspase; DAPI, 4',6-diamidino-2-phenylindole; FL, full length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSDM-D, gasdermin D; IL, interleukin; KO, knockout; RFU, relative fluorescent unit; RIPK3, receptor-interacting serine/threonine kinase 3; WT, wild-type.

Fig 3: The effect of ΔeseB-D on Casp4 activation.(A) Differentiated THP-1 (dTHP-1) cells were treated with wild-type (WT) Edwardsiella tarda or the ΔeseB-D mutant for 2 hr or 4 hr. The cells were subjected to cell death analysis (A) and immunoblot with antibodies against Casp4 and β-actin (B). Data in panel A are the means of triplicate assays and are shown as means ± SD. Figure 2—figure supplement 1—source data 1.PDF file containing the original blots for Figure 2—figure supplement 1. Figure 2—figure supplement 1—source data 2.Original files for blots are displayed in Figure 2—figure supplement 1. Figure 2—figure supplement 1—source data 3.The numerical source data corresponds to Figure 2—figure supplement 1.

Fig 4: The ability of Edwardsiella tarda to induce pyroptosis in human macrophages.(A) The schematic of experimental design. (B–E) Differentiated THP-1 (dTHP-1) cells were infected with E. tarda for the indicated hours and then subjected to microscopy (B), measurement of cell death (C), IL-1β release (D), and Western blot (E) using antibodies against Casp1, GSDMD, and β-actin (loading control). In (B), red arrowheads indicate pyroptotic cells; scale bar, 10 μm. (F–I) dTHP-cells in the form of wild-type (WT), knockout (KO) variants (Aim2-KO, NLRC4-KO, ASC-KO, Casp4-KO, and GSDMD-KO), and knockdown (KD) variants (NLRP3-KD and Casp1-KD) were infected with or without E. tarda for 2 or 4 hr, and then assessed for cell death (F, H) and IL-1β release (G, I). For panels C, D, and F-I, data were the means of triplicate assays and shown as means ± SD. ns, not significant, ***p<0.001, ****p<0.0001, one-way ANOVA with Dunnett’s multiple-comparison test. Figure 1—source data 1.PDF file containing the original blots for Figure 1E. Figure 1—source data 2.Original files for blots are displayed in Figure 1E. Figure 1—source data 3.The numerical source data corresponds to Figure 1.