Fig. 4. RIPK3 regulates SARS-CoV-2-induced necroptosis and inflammatory signaling.

a, b Calu-3 cells stably expressing non-targeting shRNA (scramble) or shRNAs against RIPK3 were infected with SARS-CoV-2 at MOI of 0.1. Cells and supernatants were harvested at 48 hpi. a NP and P17 levels in supernatants and expression levels of RIPK3, pMLKL, PARP1, Casp3, cleaved-Casp3, Pro-IL-1β or NP in cell lysates were determined by western blot assay. b Relative mRNA level of indicated cytokine and chemokine genes was detected by qRT-PCR. c, d Calu-3 cells stably expressing non-targeting sgRNA (scramble) or sgRNAs against MLKL were infected with SARS-CoV-2 (MOI = 0.1). Cells and supernatants were harvested at 48 hpi. c NP and P17 levels in supernatants and expression levels of MLKL, pMLKL, PARP1, Casp3, cleaved-Casp3, Pro-IL-1β or NP in cell lysates were determined by western blot assay. d Relative mRNA level of indicated cytokine and chemokine genes was detected by qRT-PCR. e, f Calu-3 cells were pre-treated with GSK872 at concentration of 1, 5 or 10 μM followed by infection with SARS-CoV-2 (MOI = 0.1) for 48 h. e NP and P17 level in the supernatants, pMLKL, pro-IL-1β or NP in cell lysates were measured by western blot assay. f Relative mRNA level of indicated cytokine and chemokine genes was determined by qRT-PCR. g, h ZBP1 knockout Calu-3 cells reconstituted with FLAG-tagged full-length ZBP1or truncation mutants deleted of Zα2 domain (ZBP1-∆Zα2) or RHIM domain (ZBP1-∆RHIM) were infected with SARS-CoV-2 (MOI = 0.1) or mock treated. g Cell lysates were harvested at 72 hpi and immunoprecipitated with anti-Flag magnetic beads, and detected by western blot assay. h Cell lysates and supernatant were harvested at 48 hpi. IL-1β P17 levels in the supernatants and the expression level of pMLKL, ZBP1or NP in the cell lysates were determined by western blot analysis. Data shown are means ± SEM. Statistical significance was analyzed by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.