Fig. 5. The Z-DNA binding domain 2 (Zα2) of ZBP1 is critical for mediating tumor necroptosis.

a Immunoprecipitation and western blotting analysis representative of three independent experiments. MVT-1 cells stably overexpressing HA-ZBP1 were treated with 5-Aza-2′-deoxycytidine (5-AD), for 3 days followed by glucose deprivation (GD) condition for the indicated time points. Cell lysates were immunoprecipitated with HA antibody and the immunoprecipitated complexes were immunoblotted. b Structure of mouse ZBP1 protein indicating the strategy for RHIM A mutant or Zα2 mutant (left panel). Western blot analysis representative of three independent experiments of MVT-1 ZBP KO cells transfected with reconstituted WT ZBP1 or RHIM A mutant or Zα2 mutant. The transfected cells were then treated with 5-AD for 3 days, followed by GD condition for 24 h, by using the indicated antibodies (right panel). c FVB/NJ mice were implanted with the MVT-1 CRISPR CT or MVT-1 ZBP1 KO or ZBP1 KO reconstituted with WT ZBP1 or RHIM A mutant or Zα2 mutant protein. Representative images of H&E and immunohistological stained sections with phospho-MLKL (p-MLKL) or cleaved caspase 3 (cl.Casp-3) antibody of each tumors. Scale bar, 50 µm. d Western blotting analysis representative of three independent experiments of 5-week tumor lysates from mice implanted as in Fig. c. The tumor lysates were determined by using the indicated antibodies. e FVB/NJ mice were implanted with the MVT-1 ZBP1 KO cells reconstituted with WT ZBP1 or Zα2 mutant protein. Left panel shows the representative images of H&E stained lung sections from mice showing lung metastasis. Scale bar, 2 mm. Right panel shows the quantification of the metastatic foci in lungs from mice at 5-week post-implantation. Data are presented as mean values ± SEM. Two-sided student’s t test was used to determine the statistical significance of differences between groups. Differences with P values < 0.05 were considered significant. Source data are provided as a Source Data file.