S161E mutation sensitizes the cecum to TNF-induced damage. (A–C) Genetic analyses of offspring from intercrosses of Ripk1^S161E/+, Ripk1^S161N/+, and Ripk1^S161A/+ parents, respectively. (D) Primary BMDMs from Ripk1^+/+, Ripk1^S161N/S161N, or Ripk1^S161E/S161E mice (three littermate 8- to 12-wk-old male mice of each genotype) were treated with TNF (10 ng/ml) + zVAD (20 μM), TNF (20 ng/ml) + SMAC mimetic (1 μM) + Emricasan (5 μM), or SMAC mimetic (1 μM) + Emricasan (5 μM) for the indicated time points with or without Nec-1s (10 μM). Cell survival was measured. Data are presented as mean ± SD. Cell lysates of nontreated primary BMDMs were analyzed by immunoblotting to detect proteins as indicated. (E) Representative H&E staining images of ceca from Ripk1^+/+ mice after TNF treatment for 6 h (400 μg/kg, i.v.) or PBS. Three 8- to 12-wk-old littermate male mice were used for each treatment. Scale bars, 100 μm. (F) Representative H&E staining images of livers, spleens, lungs, and kidneys from Ripk1^S161E/S161E mice after PBS or TNF treatment for 6 h (50 μg/kg, i.v.). Three 8- to 12-wk-old littermate male mice were used for each treatment. Scale bars, 100 μm. (G) 6- to 8-wk-old male littermate mice were treated with or without cecectomy and then injected with TNF after 4 wk (50 μg/kg i.v.). Survival curve is presented as a Kaplan–Meier plot, and the log-rank (Mantel–Cox) test (two-sided) is performed to determine statistical significance. ns, P ≥ 0.05. Data of body temperature are presented as mean ± SD. The above experiments were independently performed twice. Source data are available for this figure: SourceData FS1.