Fig 2: The effect of the PARP1 inhibitor PJ34 on inflammation and cell death caused by STING activation. A, B Western blot analysis of STING pathway and PAR level in RAW264.7 (A) or iBMDM (B) cells treated with DMXAA alone or in combination with PARP1 inhibitor (PJ34) for the indicated times (hours). C Evaluate the activation of the IFN luciferase reporter gene in RAW264.7 cells mediated by ADU, with or without PARP1 inhibitors (PJ34, 3AB, Olaparib) or NAC. D Relative mRNA expression levels of cytokines (IFN-β, IL-6, TNF-α, CCL2, ISG15, IL-10, IL-1β, and NOS2) in RAW264.7 cells treated with DMXAA alone or in combination with PJ34. E Immunofluorescence staining of RAW264.7 cells treated with DMXAA for 12 hours, with or without PJ34, using Calcein AM (green, live cells) and PI (red, dead cells). Scale bar = 25 μm. F Quantification of cytotoxicity and cell viability after DMXAA treatment with or without PJ34 for the indicated times. Cytotoxicity was assessed by LDH release, and cell viability by CCK-8 assay. G Subcellular fractionation and immunoblotting showing AIF localization in cytoplasmic and nuclear fractions after DMXAA treatment with or without PJ34. H Quantification of AIF protein levels in cytoplasmic and nuclear fractions. I immunofluorescence images of AIF (green) and nuclei (DAPI, blue) in RAW264.7 cells after DMXAA treatment with or without PJ34. Scale bar = 5 μm. Data were shown as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Fig 3: ERO1 interacts with the CTT domain of STING and is recruited to the STING complex. A Structural modeling of the STING-ERO1 complex. Predicted interactions between ERO1 and STING monomer or dimer. B, C Co-immunoprecipitation (Co-IP) assays showing interaction between ERO1 and STING. HEK293T cells were co-transfected with Myc-ERO1 and Flag-STING, followed by IP with anti-Myc (B) or anti-Flag (C) and immunoblotting. D Immunofluorescence imaging of STING (red), ERO1 (green), and nuclei (DAPI, blue) in RAW264.7 cells treated with DMXAA for 1 h or 3 h. Scale bar = 5 μm. E Schematic of full-length and domain-deletion STING mutants. TM: transmembrane; DD: dimerization domain; CBD: CDN-binding domain; CTT: C-terminal tail. F, G Co-IP assays showing the interaction of ERO1 with various STING domain-deletion mutants. HEK293T cells were transfected with Myc-ERO1 and Flag-tagged full-length or truncated STING constructs, followed by IP with anti-Myc (F) or anti-Flag (G) and immunoblotting

Fig 4: Role of STING in regulating PARP1 activation and tissue damage caused by sepsis. A Representative HE staining images of intestinal and lung tissue after CLP in wild-type and STING knockout mice. Scale bar = 50 μm. B TUNEL staining indicated the presence of dead cells (green fluorescence) in the intestinal tissue of each group. Scale bar = 50 μm. C Quantitative assessment of intestinal histopathological damage scores (chiu’s score) after CLP in wild-type and STING knockout mice. D Relative mRNA expression levels of IL6 in intestinal tissues of each group. E Western blot analysis and quantification of PARP1, PAR and STING protein expression in intestinal tissues. F, G Representative HE staining images of intestinal and lung tissue after CLP in wild-type, STINGLysM⁻/⁻ and STINGVil⁻/⁻ mice. Scale bar = 50 μm. H Quantitative assessment of intestinal histopathological damage scores (chiu’s score) in each group. I, J Relative mRNA expression levels of IL6, TNF-α, IL-1β, CCL2 and ISG15 in intestinal tissues of each group. K, L Western blot analysis and quantification of PARP1, PAR, AIF and MIF protein expression in intestinal tissues of each group. Data were shown as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Fig 5: STING activation leads to DNA damage, AIF translocation, and parthanatos.A Immunoblotting analysis of STING signaling pathway in RAW264.7 cells treated with DMXAA or ADU for the indicated times (hours). B PAR polymer formation following treatment with DMXAA or ADU, as assessed by immunoblotting for PAR and β-actin. C Relative mRNA expression levels of inflammatory cytokines and signaling genes (IL-6, IL-1β, IFN-β, TNF-α, CCL2, IL-10, STAT1, STING, MIF, and AIF) in RAW264.7 cells treated with DMXAA or ADU for the indicated times. D Western blot analysis of STING, TBK1, γH2AX, and PAR levels in RAW264.7 cells treated with DMXAA for indicated times. E Representative immunofluorescence images showing γH2AX foci formation (red) and nuclear staining (DAPI, blue) in control and DMXAA-treated RAW264.7 cells. Scale bar = 10 μm. F Quantification of cytotoxicity and cell viability after DMXAA treatment at various time points. Cytotoxicity was assessed by LDH release, and cell viability by CCK-8 assay. G Subcellular fractionation and immunoblotting showing AIF localization in cytoplasmic and nuclear fractions after DMXAA treatment. GAPDH and Lamin B1 served as loading controls for cytoplasmic and nuclear fractions, respectively. H Quantification of AIF protein levels in cytoplasmic and nuclear fractions. I Representative immunofluorescence images of AIF (red) and nuclei (DAPI, blue) in control and DMXAA-treated RAW264.7 cells showing translocation of AIF to the nucleus. Scale bar = 5 μm. Data were shown as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001