Fig 2: ZBP1 promotes CASP8-mediated cell death and IL-1β release.a Schematic of protein domains in TRIF, RIPK1, and ZBP1. b, c Cell death over time in indicated BMDMs stimulated with LPS/5z7 as measured by (b) propidium iodide (PI) incorporation or (c) LDH release. d Cell death over time as measured by PI incorporation in B6 and Zbp1−/− BMDMs treated as indicated. e Full-length and cleaved products of indicated caspases and GSDMD from whole-cell lysates (WCL) or precipitated from the supernatant (Sup.) of B6 or Zbp1−/− BMDMs stimulated with LPS/5z7 for 1–6 h. f Levels of total, pRIPK1 (S166), and pRIPK1 (S321) in B6 or Zbp1−/− BMDMs stimulated with LPS or LPS/5z7 for 1–3 h. g Full-length and cleaved products of CASP1 and IL-1β from whole-cell lysates of B6 and Zbp1−/− BMDMs stimulated as indicated for 1–3 h. h IL-1β release after 6 h and (i) percentage of ASC+ cells after 2 h in B6, Zbp1−/−, and Ripk3−/−Casp8−/− BMDMs stimulated as indicated. Data from cell death assays and western blots are representative of 3 or more biologically independent experiments, cell death data are presented as the mean ± SD of triplicate wells, n = 10,000 cells examined in three individual wells. IL-1β release, ASC percentage, and qPCR data are presented as the mean ± SD for triplicate wells from n = 4 biologically independent experiments. Two-way analysis of variance (ANOVA) was used for comparison between groups. Source data for all experiments are provided as a Source data file. See also Supplementary Figs. 2 and 3.

Fig 3: The thioredoxin-interacting protein (TXNIP)-inflammasome-caspase-1 axis regulates the clearance of bacteria from macrophages. (A) The levels of interleukin (IL)-1β in conditioned media from wild-type (WT) and Txnip knockout (KO) mouse peritoneal macrophages after treatment with E. coli for the indicated times. Data are expressed as the mean ± SD (***P < 0.001 compared with WT). (B) The protein expression levels of caspase-1 P45 and caspase-1 P10 in mouse WT and Txnip KO mouse peritoneal macrophages after treatment with E. coli for the indicated times. (C) Caspase-1 activity, with or without ZVAD treatment, in mouse WT and Txnip KO mouse peritoneal macrophages 6 h after the treatment with E. coli. Data are expressed as the mean ± SD (***P < 0.001 compared with WT). (D) Representative images of bacteria-laden WT and Txnip KO mouse macrophages 6 h after a 1-h treatment with GFP-expressing E. coli with or without ZVAD treatment and removal of extracellular bacteria. Scale bar, 10 μm.

Fig 4: Transcriptional profiling of the liver in NASH mice and verification of NASH associated signaling pathways. A Transcriptional profiling of the liver tissue of C5aR1−/− and WT mice. B KEGG pathway analysis. C Toll-like receptor signaling pathway associated genes were showed in heatmap. D The mRNA level of Tlr4 was measured by qRT-PCR. The data is representative of three independent experiments. E The protein levels of TLR4, p-NFκB P65 and NFκB P65. F The mRNA level of Nlrp3 was determined by qRT-PCR. G The protein levels of NLRP3 and CASP1. H RAW264.7 cells were treated with the recombinant protein C5a (50 ng/mL) for 24 h. The protein levels of TLR4, NLRP3, and CASP1 were determined by western blot. I The protein levels of iNOS, CD86, CD163 and CD206 were measured by Western blot. J The mRNA levels of iNOS and CD163. K. Protein levels of p-AKT and AKT. Experiments were repeated three times. Results are presented as mean ± SD. ***P < 0.001. n = 6 per group

Fig 5: miR-369-3p reduced the activation and release of caspase-1. (A) Western blot analysis of pro-CASP1 protein expression after miR-369-3p mimic transfection following LPS and Nigericin stimulations in the BMDM cell line. To normalize data, GAPDH was used as housekeeping protein. Raw data of the independent experiments of Western blot were reported in Supplementary File S1. (B) Caspase-1 activity after LPS and Nigericin stimulations in the mimic transfected BMDM cell line. (C) In vitro cell viability assay of BMDM cells transiently transfected with miR-369-3p mimic and stimulated with LPS and Nigericin. (* p < 0.05; ** p < 0.01; *** p < 0.0001).