Extended Data Fig. 3: CASP8(C362S) induces necroptosis-independent tissue destruction.
From: Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis
a, Genotyping PCR of respective endothelial cells (ECs) after treatment with cell-permeable recombinant HTNCre protein. Results are representative of two individual experiments. b, Analysis of caspase-8 and caspase-3 processing by western blot after treatment with TNF (10 ng ml−1), CHX (2.5 µg ml−1) or both (TNF and CHX). Results are representative of two individual experiments. c, Top, viability of endothelial cells after treatment with TNF (10 ng ml−1), Nec-1 (30 µM) or both (TNF and Nec-1) for 6 h (top) in biologically independent replicates. Dots represent individual biological replicates (n = 3). Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis. n = 3, representative of two individual experiments. Bottom, western blot analysis of cell lysates examining phosphorylated MLKL (P-MLKL) and β-actin. Results are representative of two individual experiments. d, Expected, observed and weaned numbers of mice per genotype obtained from the indicated crossings. e, Spleen weight and spleen:body weight ratios of 8- and 15-week-old mice of the indicated genotypes. Dots and circles, individual mice. Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis compared to the corresponding Casp8WT/WT values. f, Representative images of spleen, axial and inguinal lymph nodes (LN) and mesenteric lymph nodes (left) as well as splenic sections from Casp8WT/WTRipk3−/− (n = 3), Casp8C362S/WTRipk3−/− (n = 3) and Casp8C362S/C362SRipk3−/− mice (n = 3) stained with H&E (right) from 15-week-old mice. Scale bars, 300 µm. g, Cardiac blood was analysed for cell numbers, and haematocrit and haemoglobin concentrations from 8–15-week-old mice. Dots and circles, individual mice. Data are mean ± s.e.m. One-way ANOVA followed by Dunnett’s post-analysis compared to the corresponding Casp8WT/WTRipk3−/− values. Exact P values (from left to right): erythrocytes, P = 0.9774, P = 0.0607; haematocrit: P = 0.9961, P = 0.0462; lymphocytes, P = 0.5812, P = 0.2426; monocytes, P = 0.9944, P = 0.0693; neutrophils, P = 0.4614, P = 0.4349; haemoglobin, P = 0.8326, P = 0.0025. h, Expected, observed and weaned numbers of embryos (left) and mice (right) per genotype obtained from the indicated crossings.
