Fig. 7

Z-DNA binding protein 1 (ZBP1)-activated receptor-interacting protein kinase 3 (RIPK3) is required for pulmonary MPVECs PANoptosome. a Immunofluorescence images of wild type (WT) primary MPVECs at 36 h post co-treatment with lipopolysaccharide (LPS) and recombinant mouse CIRP (rmCIRP) show significant co-localization of ZBP1 (green) and RIPK3 (red) (scale bar = 20 μm). The arrows from “i” to “ii” highlight the region analyzed for colocalization analysis. The images on the right show the analysis of ZBP1 and CIRP co-localization in immunofluorescence and Pearson’s correlation coefficient calculations. b Proximity ligation assay (PLA) revealed physical interactions between ZBP1 and RIPK3 as red spots in WT primary MPVECs 36 h after co-treatment with LPS and rmCIRP (scale bar = 20 μm). c Representative images of cell death and real-time analysis of cell death in WT, Zbp1^−/−, and Ripk3^−/− primary MPVECs co-treated with LPS and rmCIRP (scale bar = 100 μm). d Immunoblot analysis of ZBP1, RIPK3, ASC, AIM2, CASP8, and CASP3 following immunoprecipitation (IP) with anti-ZBP1 or control IgG antibodies in WT, Zbp1^−/−, and Ripk3^−/− primary MPVECs after 36 h of co-treatment with LPS and rmCIRP. e Hematoxylin and eosin (H&E)-stained lung tissues from WT, Zbp1^−/−, and Ripk3^−/− mice at 36 h post-CLP (scale bar = 50 μm), arrows indicate lung injury characterized by alveolar septal thickening and inflammatory cell infiltration. A corresponding quantification of histology scores is provided below. f Survival analysis of male WT, Zbp1^−/−, and Ripk3^−/− mice aged 6–8 weeks following CLP (n = 10) revealed significantly different survival rates in Zbp1^−/− and Ripk3^−/− mice compared to WT mice. Data are presented as mean ± SD. ^**P < 0.01, ^***P < 0.001. MPVEC mouse pulmonary vascular endothelial cell, ASC apoptosis-associated speck-like protein containing a CARD, AIM2 absent in melanoma 2, CASP8 caspase-8, CASP3 caspase-3