Fig 2: IFN-ß–driven lethality, cytokine storm and cell death depend on ZBP1 during ß-coronavirus infection.(A) Volcano plot showing the genes that are enriched or depleted in immortalized bone marrow-derived macrophages (iBMDMs) following a genome-wide CRISPR/CAS9 knockout screen of cell death induced by mouse hepatitis virus (MHV) infection (MOI 0.2, 24 hours). (B) Survival of 6- to 12-week-old wild type (WT) and Zbp1 –/– mice treated with PBS (n = 15 for WT and n = 16 for Zbp1 –/– mice) or IFN-ß (n = 21 for WT and n = 15 for Zbp1 –/– mice) on day 1 and 3 after intranasal infection with MHV. (C) Immunoblot analysis of ZBP1 in the lung samples from PBS- or IFN-ß–treated WT mice 3 days after MHV infection. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Actin was used as the internal control. (D) Real-time analysis of cell death in MHV-infected WT or Zbp1 –/– BMDMs in the presence or absence of IFN-ß. (E) Representative images of cell death in media- or IFN-ß–treated WT or Zbp1 –/– BMDMs are shown at 24 hours after MHV infection. Scale bar, 50 µm. (F) Real-time analysis of cell death in MHV-infected WT or Zbp1 ?Za2 BMDMs in the presence of IFN-ß. (G) Real-time analysis of cell death in influenza A virus (IAV)-infected WT or Zbp1 –/– BMDMs in the presence and absence of IFN-ß. (H) Survival of 6- to 12-week-old WT (n = 15) and Zbp1 –/– (n = 14) mice after intranasal infection of IAV. (I) Survival of 6- to 12-week-old WT and Zbp1 ?Za2 mice treated with PBS (n = 29 for WT and n = 21 for Zbp1 ?Za2 mice) or IFN-ß (n = 28 for WT mice) on day 1 and 3 after intranasal infection of IAV. Survival data are pooled from 2 infection experiments (B, H, I). All other data are representative of at least three independent experiments. *P < 0.05, ***P < 0.001 and ****P < 0.0001. Analysis was performed using the one-way ANOVA (D, G), two-tailed t test (F) or log-rank test (Mantel-Cox) (B, H, I). Data are shown as mean ± SEM.

Fig 3: ZBP1 is highly increased in late stage of mouse and human tumors.a–e Western blotting analysis representative of three independent experiments. a MVT-1 tumors were collected at 2–5 weeks post-implantation and tumor cell lysates were analyzed by western blotting with the indicated antibodies. b MMTV-PyMT breast tumors were collected at 10–15 weeks and tumor cell lysates were analyzed by western blotting with the indicated antibodies (*, non-specific band). c GFP-MVT-1 tumor cells were collected at 5-week post implantation and the lysates were analyzed by western blotting with the indicated antibodies. d C57BL/6 J mice implanted with syngeneic B16 mouse melanoma cells and tumor cells were collected at 2-weeks post-implantation. Western blot analysis of cultured B16 cell lysates or B16 tumor cell lysates was done using the indicated antibodies. e BALB/c-nu/nu mice implanted with MCF7 human breast cancer cells and tumor cells were collected at 8-weeks post-implantation. Western blot analysis of cultured MCF7 cell lysates or MCF7 tumor cell lysates was done using the indicated antibodies. To be comparable to the late stage necrotic tumors, the tumors from these models in d and e were collected when they approached 1500–2000 mm3 in volume and had tumor necrosis. f Quantitative real-time PCR analysis of the relative expression of Ripk1 or Zbp1 or Ripk3 mRNA from cultured MVT-1 cells or MVT-1 tumor cells isolated from the mice implanted with MVT-1 cells and collected at 5 weeks (n = 4, each). Data are presented as mean values ± SEM. g Differential expression of human ZBP1 was analyzed for human breast cancer dataset (TCGA-BRCA) and performed for each tumor stage (Normal stage, n = 112; Stage I, n = 182; Stage II, n = 627; Stage III, n = 249; Stage IV, n = 20). Data are presented as mean values ± SD. h Methylation-specific PCR of genomic DNA from MVT-1 or 5-Aza-2'-deoxycytidine (5-AD), treated MVT-1 or 5-week MVT-1 tumor was detected by using methylation primer (M) or unmethylation primer (UM) or regular primer (R) for RIPK3. The PCR result is representative of three independent experiments. 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.

Fig 4: ABT-737 induces cancer cell necrosis and inhibits cell viability and invasion when the RIP1 gene is knocked down in bladder cancer cells. (A) Expression levels of RIP1 in UMUC3 and 5637 cells were analyzed via western blotting after treatment with RIP1 siRNA for 12 h, and the results were (B) semi-quantified. (C) Protein expression levels of HMGB1, ZBP1, RIP3 and MLKL were analyzed in RIP1-KD UMUC3 and RIP1-KD 5637 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. The results were semi-quantified in (D) UMUC3 and (E) 5637 cells. (F) Transwell invasion assays were performed with RIP1-KD UMUC3 cells. (G) Quantification of Transwell assay results (H) MTT assays were performed to examine the viability of RIP1-KD UMUC3 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. (I) Transwell invasion assays were performed with RIP1-KD 5637 cells. (J) Quantification of Transwell assay results. (K) MTT assays were performed to examine the viability of RIP1-KD 5637 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. *P<0.05 vs. control or siRNA NC. OD, optical density; scale bar=50 µm; ZBP1, Z-DNA binding protein 1; RIP, receptor-interacting protein; HMGB1, high mobility group box 1; MLKL, mixed-lineage kinase domain-like protein; siRNA, small interfering RNA; NC, negative control; KD, knockdown.

Fig 5: RIP3 serves a major role in the cell necrosis induced by ABT-737 treatment. (A) Expression levels of RIP3 in UMUC3 and 5637 cells after treatment with RIP3 siRNA for 12 h were analyzed via western blotting, and the results were (B) semi-quantified. (C) Protein expression levels of HMGB1, ZBP1 and MLKL were analyzed in RIP3-KD UMUC3 and RIP3-KD 5637 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. The results were semi-quantified in (D) UMUC3 and (E) 5637 cells. (F) Transwell invasion assays were performed with RIP3-KD UMUC3 cells. (G) Quantification of Transwell assay results. (H) MTT assays were performed to examine the viability of RIP3-KD UMUC3 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. (I) Transwell invasion assays of RIP3-KD 5637 cells. (J) Quantification of Transwell assay results. (K) MTT assays were performed to examine the viability of RIP3-KD 5637 cells treated with Z-VAD-FMK combined with ABT-737 for 12 h. (scale bar=50 µm) *P<0.05 vs. siRNA NC or control. OD, optical density; ZBP1, Z-DNA binding protein 1; RIP, receptor-interacting protein; HMGB1, high mobility group box 1; MLKL, mixed-lineage kinase domain-like protein; siRNA, small interfering RNA; NC, negative control; KD, knockdown.