Fig 2: The RIPK1-FADD interaction negatively controls RIPK3-mediated lethal inflammation(A) Expected Mendelian ratios and observed numbers of offspring of Ripk1RE/WT Mlkl−/− intercrosses (n = 400).(B) Observed numbers of offspring of Ripk1RE/WT Mlkl−/− intercrosses at birth (present at post-natal day 3 [P3]) and at weaning (P21).(C) Post-natal survival up to 200 days of Ripk1WT/WT Mlkl−/− (n = 13), Ripk1RE/WT Mlkl−/− (n = 13), and Ripk1RE/RE Mlkl−/− (n = 13) animals.(D and E) H&E staining of colon (D) or skin (E) tissue of Ripk1WT/WT Mlkl+/+ (top) and Ripk1RE/RE Mlkl−/− (bottom) mice. Magnified areas (4×, scale bar: 200 μm; 10×, scale bar: 100 μm; and 40×, scale bar: 20 μm, respectively) are indicated. Images represent 2 independent experiments.(F) Selected Hallmarks pathway enrichment score plots of RNA sequencing gene set enrichment analysis comparing liver transcriptomes of Ripk1RE/WT Mlkl−/− and Ripk1RE/RE Mlkl−/− mice. Calculation and analysis are described in the STAR Methods.(G) Expected Mendelian ratios and observed numbers of offspring of Ripk1RE/WT Fadd−/+ Mlkl−/− intercrosses (n = 196).(H) Post-natal survival up to 200 days of Ripk1WT/WT Fadd+/+ Mlkl−/− (n = 5), Ripk1RE/WT Fadd−/− Mlkl−/− (n = 5), and Ripk1RE/RE Fadd−/− Mlkl−/− (n = 9) animals.See also Figure S3.

Fig 3: ZBP1 drives RIPK3-mediated death of Ripk1RE/RE and Ripk1REKA/REKA embryos(A) WT, Ripk1−/−, or Ripk1−/− Mlkl−/− MEFs reconstituted with RIPK1 (WT), RIPK1-K45A (K45A), RIPK1-R588E (RE), or RIPK1-R588E/K45A (REKA) were analyzed by immunoblotting using indicated antibodies. Representative of two independent experiments.(B) Tissues from 4- to 6-week-old Ripk1WT/WT (WT), Ripk1RE/RE Ripk3−/− (RE), or Ripk1REKA/REKA Ripk3−/− (REKA) mice were examined by immunoblotting using indicated antibodies. Representative of two independent mice.(C) Expected Mendelian ratios and observed numbers of offspring of Ripk1RE/WT Zbp1−/− intercrosses (n = 137).(D) Expected Mendelian ratios and observed numbers of offspring from Ripk1REKA/WT Zbp1−/− intercrosses (n = 125).(E) Post-natal survival up to 200 days of Ripk1WT/WT Zbp1−/− (n = 5), Ripk1RE/WT Zbp1−/− (n = 5), and Ripk1RE/RE Zbp1−/− (n = 5) mice.(F) Post-natal survival up to 200 days of Ripk1WT/WT Zbp1−/− (n = 7), Ripk1REKA/WT Zbp1−/− (n = 7), and Ripk1REKA/REKA Zbp1−/− (n = 7) mice.See also Figure S4.