Fig. 5

Z-DNA binding protein 1 (ZBP1) regulates pulmonary vascular endothelial cell (PVEC) PANoptosis in response to lipopolysaccharide (LPS) and extracellular CIRP (eCIRP). a Immunoblot analysis of ZBP1 in WT and Zbp1^−/− primary MPVECs after treatment with LPS alone, rmCIRP alone, or co-treatment with LPS and rmCIRP for 48 h. b Representative images of cell death and quantification showing the percentage of cell death in WT, Zbp^−/− primary MPVECs treated with LPS and rmCIRP for 48 h (scale bar = 100 μm). c Immunoblot analysis of pro-activated (P45) and activated (P20) CASP1; pro-activated (P53), activated (P30), and inactivated (P20) GSDMD; and AIM2; pro-cleaved (P35) and cleaved (P17) CASP3, pro-cleaved (P55) and cleaved (P18) CASP8; p-MLKL and MLKL in WT and Zbp1^−/− MPVECs at 0 h or co-treatment with LPS and rmCIRP for 48 h. d Immunofluorescence images of WT and Zbp1^−/− primary MPVECs 48 h after co-treatment with LPS and rmCIRP (scale bar = 5 μm). Arrowheads indicate the PANoptosome. The bar chart displays the quantification of the percentage of cells with ASC⁺CASP8⁺RIPK3⁺ specks among the ASC speck⁺ cells. The data are presented as mean ± SD. ^***P < 0.001. rmCIRP recombinant mouse cold-inducible RNA-binding protein, MPVEC mouse pulmonary vascular endothelial cell, GSDMD gasdermin D, CASP1 caspase-1, AIM2 absent in melanoma 2, CASP3 caspase-3, CASP8 caspase-8, p-MLKL phosphorylated mixed lineage kinase domain-like protein, MLKL mixed lineage kinase domain-like protein, RIPK3 receptor-interacting protein kinase 3, ASC apoptosis-associated speck-like protein containing a CARD