IEC necroptosis triggers TNF-induced death of Ripk1 ^S161E/S161E mice. (A and B) Survival curves and body temperature of 14-wk-old male littermate bone marrow–transplanted Ripk1^+/+, Ripk1^S161E/S161E, or Ripk1^S161N/S161N mice after injection with TNF (50 μg/kg i.v. in A, 200 μg/kg i.v. in B). Mouse survival is presented as a Kaplan–Meier plot, and the log-rank test is performed. ns, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Data of body temperature are presented as mean ± SD. (C) Survival curves and body temperature of 8- to 12-wk-old male littermate mice of the indicated genotypes after TNF injection (50 μg/kg, i.v.). (D) Mice (n = 6) of the indicated genotypes were i.v. injected with TNF (50 μg/kg). At the indicated time points, serum samples were collected for measurements of LDH release. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. ****P < 0.0001. (E) Representative H&E staining images of ceca from three mice of each indicated genotype i.v. treated with 50 μg/kg of TNF for 6 h. Scale bars, 100 μm. (F) Representative immunofluorescence staining images of ceca from five mice of each indicated genotype after TNF treatment for 6 h (50 μg/kg, i.v.). E-cadherin (E-Cad, red), phospho-MLKL S345 (p-MLKL, green), Ki-67 (purple), phospho-RIPK3 T231 + S232 (p-RIPK3, light blue), and Hoechst (blue). Scale bars, 50 μm. Percentages of p-MLKL⁺ cells in E-Cad⁺ cells and p-RIPK3⁺ cells in Ki-67⁺ cells were quantified using ImageJ. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. ****P < 0.0001. All mice were 8- to 12-wk-old male littermates (D–F). The above experiments were independently performed twice.