Fig 2: Reversal of TRIM13 deficiency-induced pyroptosis and DC activation by STING inhibition. (a) Scheme for STING inhibitor administration before CLP. (b-e) Trim13 cKO mice were intraperitoneally injected with the STING inhibitor C-176 (750 nmol in 200 μl of corn oil) 0.5 h prior to CLP. Survival data were collected for analysis. Splenic DCs were isolated at the indicated post-CLP time points for subsequent analyses. (b) Survival curves following CLP (n = 20 mice per group), demonstrating the impact of C-176 on the outcomes of WT and Trim13 cKO mice. (c) Flow cytometry analysis of splenic DC pyroptosis rate at the indicated post-CLP time points (n = 3 mice per group). DCs were stained for CASP-1 (FAM-FLICA) and 7-AAD. Representative flow cytometry plots are shown on the left, with quantitative data presented on the right. (d) Flow cytometry analysis of the expression levels of CD80, CD86, and MHC II in splenic DCs (n = 3 mice per group). The results are displayed below. (e) the proportion of divided CD4+ T cells stained for CFSE was evaluated using flow cytometry (n = 3 mice per group). The quantitative results are shown on the right. Data are expressed as means ± SD. ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001. DCs dendritic cells, SD standard deviation, WT wild-type, TRIM13 tripartite motif 13, CLP cecal ligation and puncture

Fig 3: TRIM13 silencing enhances p-IRF3 nuclear translocation and upregulates NLRP3 expression in DCs following sepsis. (a) Representative confocal microscopy images showing the nuclear translocation of p-IRF3 (green) in con and Trim13 KD DC2.4 cells after LPS stimulation (1 μg/ml, 24 h). DAPI: Blue; ER-tracker: Red; scale bar: 20 μm. (b) qPCR analysis was performed to assess NLRP3 mRNA expression in con and Trim13 KD DC2.4 cells stimulated with LPS (1 μg/ml, 24 h), DMSO, ESI (10 μm, 4 h), XRK3F2 (10 μm, 24 h) or GSK-690693 (15 μm, 24 h). Data are normalized to GAPDH expression as an internal control and presented as the relative fold change using the ΔΔCt method. Data are expressed as means ± SD. ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (c) Co-IP was used to assess the interaction between NLRP3 and STING in con and Trim13 KD DC2.4 cells stimulated with LPS (1 μg/ml, 24 h). Cell lysates were immunoprecipitated with an anti-STING antibody and probed with an anti-NLRP3 antibody. IgG controls were included to confirm specificity. (d) Immunoblot analysis of WT and Trim13Cd11c DCs revealed protein expression associated with GSDMD-dependent pyroptosis after various stimulation (LPS, 1 μg/ml, 24 h; DMSO, 24 h; ESI, 10 μm, 4 h; XRK3F2, 10 μm, 24 h; C-176, 10 μm, 1 h pretreatment) (n = 3). The quantitative results are displayed on the left and below. DCs dendritic cells, ER endoplasmic reticulum, SD standard deviation, TRIM13 tripartite motif 13, STING stimulator of interferon genes, LPS lipopolysaccharide

Fig 4: TRIM13 deficiency in splenic DCs alters ER morphology and affects the outcomes of septic mice. (a) Immunoblot analyses revealed TRIM13 expression in primary WT splenic DCs (WT DCs) at the indicated post-CLP time points. The protein levels were normalized to those of β-actin and presented on the right (n = 3). (b) Representative images of whole spleens resected at the indicated post-CLP time points. (c) WBC counts in WT and Trim13 cKO mice at the indicated post-CLP time points (n = 5 mice per group). (d) Serum cytokine levels at the indicated post-CLP time points were quantified by ELISA (n = 5 mice per group). (e) TEM images showing ultrastructural changes in the ER morphology of WT and Trim13Cd11c DCs after stimulation with LPS (1 μg/ml) for 24 h. ER regions are highlighted in red for enhanced visualization, whereas nuclei are stained green. The quantification of the ER areas (n = 3) is shown on the right. AL, autolysosome; AP, autophagosome; EW, endoplasmic reticulum whorl; M, mitochondria; N, nucleus; scale bars: 20 μm and 5 μm. (f) Survival curves following CLP (n = 20 mice per group) demonstrating the impact of TRIM13-deficient DCs on survival outcomes. Data are expressed as means ± SD. ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001. DCs dendritic cells, ER endoplasmic reticulum, SD standard deviation, WT wild-type, TRIM13 tripartite motif 13, CLP cecal ligation and puncture, WBC white blood cell counts, ELISA enzyme-linked immunosorbent assay, TEM transmission electron microscopy

Fig 5: TRIM13 deficiency enhances gasdermin D (GSDMD)-dependent pyroptosis in splenic DCs during sepsis. (a) Time-course flow cytometry analysis of the DC pyroptosis rate following CLP. The splenic DCs were stained for CASP-1 (FAM-FLICA) and 7-AAD. Representative flow cytometry plots are shown on the left, with quantitative data displayed on the right (n = 3 mice per group). (b) Immunoblot analysis of WT and Trim13Cd11c DCs at the indicated time points post-CLP demonstrated that protein expression was related to GSDMD-dependent pyroptosis. The quantitative results are shown on the right (n = 3). (c) Concentrations of pyroptosis-related cytokines in culture supernatants from WT and Trim13Cd11c DCs were quantified by ELISA following LPS stimulation for the indicated durations (n = 3 mice per group). (d) Representative TEM images showing ultrastructural changes in WT and Trim13Cd11c DCs after stimulation with LPS (1 μg/ml) for 24 h and nigericin (nig) (20 μm) for 30 min. Nuclei are highlighted in green for enhanced visualization. N, nucleus; scale bars: 20 μm and 10 μm. Data are expressed as means ± SD. ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001. DCs dendritic cells, SD standard deviation, WT wild-type, TRIM13 tripartite motif 13, CLP cecal ligation and puncture, WBC white blood cell counts, ELISA enzyme-linked immunosorbent assay, TEM transmission electron microscopy, LPS lipopolysaccharide