Figure 6.
Cecal EC necroptosis is another trigger of TNF-induced death, and the necroptosis of IECs and ECs in the cecum of TNF-treated Ripk1S161E/S161Emice promotes each other reciprocally. (A, B, and I) 4 h after TNF treatment (50 μg/kg, i.v.), 8- to 12-wk-old male littermate mice of the indicated genotypes were injected with EB (0.5 mg/mouse). Organs were collected 20 min after EB injection, and the amount of EB extracted from the organs was measured by spectrophotometry. (C) 8- to 12-wk-old male littermate 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. (D and E) 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.). Mouse survival is presented as a Kaplan–Meier plot, and the log-rank test is performed. *P < 0.05. Data of body temperature are presented as mean ± SD. (F, H, and J) Representative immunofluorescence staining images of ceca from five mice of each indicated genotype 6 h after TNF injection (50 μg/kg, i.v.). Endomucin (red) and p-MLKL (green) in F and J; E-Cad (red) and p-MLKL (green) in H. Scale bars, 50 μm. Percentages of p-MLKL+ cells in Endomucin+ cells (F and J) or in E-Cad+ cells (H) were quantified using ImageJ. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. ****P < 0.0001. (G) Representative H&E staining images of ceca from three mice of each indicated genotype 6 h after i.v. injection with 50 μg/kg of TNF. Scale bars, 100 μm. All mice were 8- to 12-wk-old male littermates (F–H and J). All 8-wk-old male Ripk1S161E/S161ECdh5-ERT2-cre+/−Ripk3f/f mice and the littermate Ripk1S161E/S161ERipk3f/f mice in this work were orally administered with 100 mg/kg of tamoxifen for 5 consecutive days. After a 10- to 14-days break, TNF was applied. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. **P < 0.01; ***P < 0.001; ****P < 0.0001. The above experiments were independently performed twice. Refer to the image caption for details.

Cecal EC necroptosis is another trigger of TNF-induced death, and the necroptosis of IECs and ECs in the cecum of TNF-treated Ripk1 S161E/S161E mice promotes each other reciprocally. (A, B, and I) 4 h after TNF treatment (50 μg/kg, i.v.), 8- to 12-wk-old male littermate mice of the indicated genotypes were injected with EB (0.5 mg/mouse). Organs were collected 20 min after EB injection, and the amount of EB extracted from the organs was measured by spectrophotometry. (C) 8- to 12-wk-old male littermate 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. (D and E) 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.). Mouse survival is presented as a Kaplan–Meier plot, and the log-rank test is performed. *P < 0.05. Data of body temperature are presented as mean ± SD. (F, H, and J) Representative immunofluorescence staining images of ceca from five mice of each indicated genotype 6 h after TNF injection (50 μg/kg, i.v.). Endomucin (red) and p-MLKL (green) in F and J; E-Cad (red) and p-MLKL (green) in H. Scale bars, 50 μm. Percentages of p-MLKL+ cells in Endomucin+ cells (F and J) or in E-Cad+ cells (H) were quantified using ImageJ. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. ****P < 0.0001. (G) Representative H&E staining images of ceca from three mice of each indicated genotype 6 h after i.v. injection with 50 μg/kg of TNF. Scale bars, 100 μm. All mice were 8- to 12-wk-old male littermates (F–H and J). All 8-wk-old male Ripk1S161E/S161ECdh5-ERT2-cre+/−Ripk3f/f mice and the littermate Ripk1S161E/S161ERipk3f/f mice in this work were orally administered with 100 mg/kg of tamoxifen for 5 consecutive days. After a 10- to 14-days break, TNF was applied. Data are presented as mean ± SD. P values are determined by a two-tailed, unpaired t test. **P < 0.01; ***P < 0.001; ****P < 0.0001. The above experiments were independently performed twice.

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