Extended Data Fig. 1: Catalytic activity of caspase-8 is required to prevent necroptosis, but autoprocessing of caspase-8 is not required.
From: Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis
a, Schematic showing the organization of the different Casp8 alleles and of pro-caspase-8 protein. Non-coding and coding Casp8 exons are represented by grey and black boxes, respectively. b, Graph indicates cleavage of a proluminogenic caspase-8 substrate in primary MEF lysates at 6 h after treatment (−, no treatment; C, cycloheximide; T, TNF). RLU, relative luminescence unit. Circles, cells from different embryos (Mlkl−/−, n = 3; Casp8C362A/C362A Mlkl−/−, n = 3; Casp8−/− Mlkl−/−, n = 3). Bars, mean ± s.e.m. Unpaired two-tailed t-test. c, Graph indicates the percentage of primary MEFs that were viable 16 h after treatment (F, FasL; Tr, TRAIL). Circles, cells from different embryos (Mlkl−/−, n = 3; Casp8C362A/C362A Mlkl−/−, n = 2; Casp8−/− Mlkl−/−, n = 3). Bars, mean ± s.e.m. d, Western blots of primary MEFs. Each lane represents cells from a different embryo (Mlkl−/−, n = 3; Casp8C362A/C362A Mlkl−/−, n = 2; Casp8−/− Mlkl−/−, n = 3). β-Actin loading control performed after caspase-8. e, Body weights of E18.5 embryos. Circles, different embryos (Mlkl−/−, n = 11; Casp8C362A/C362A Mlkl−/−, n = 11). Lines, mean ± s.e.m. P = 0.7; unpaired two-tailed t-test. f, Kaplan–Meier survival curve of wild-type (n = 25) and Casp81×DA/1×DA (n = 29) littermates. P = 0.9; two-sided log-rank test. g, Western blots of thymocytes. The pan-caspase inhibitor emricasan revealed caspase-dependent cleavage events. Asterisk, non-specific band detected by the caspase-8 antibody. β-Actin loading control performed after cleaved caspase-8. Results representative of two independent experiments. h, Kaplan–Meier survival curve of Casp83×DA/3×DA (n = 40) and Casp84×DA/4×DA (n = 43) littermates. P = 0.2; two-sided log-rank test. i, Graph indicates the percentage of viable thymocytes before treatment (−), and after culture in either medium alone (Med.) or FasL for 24 h. Circles, cells from different mice (wild-type, n = 3; Casp81×DA/1×DA, n = 3; Casp83×DA/3×DA, n = 3; Casp84×DA/4×DA, n = 3). Bars, mean ± s.e.m. Unpaired two-tailed t-test. j, Graph indicates cleavage of a proluminogenic caspase-8 substrate in thymocyte lysates. Circles, cells from different mice (wild-type, n = 3; Casp84×DA/4×DA, n = 3). Bars, mean ± s.e.m. Unpaired two-tailed t-test. k, Graph indicates leukocyte numbers in spleen and lymph nodes (axillary, brachial, inguinal, and mesenteric) of mice aged 12–16 months (spleen, n = 3 per genotype; lymph nodes, n = 4 wild-type, n = 5 Casp81×DA/1×DA). Circles, cells from different mice. Lines, mean ± s.e.m. l, Flow cytometric analysis of lymph node cells from 1 wild-type and 2 Casp81×DA/1×DA mice aged 16 months, and as a control, one Casp8−/− Mlkl−/− mouse aged 3 months (this strain develops lymphadenopathy due to impaired Fas-induced apoptosis3). m, Graph indicates leukocyte numbers in spleen and mesenteric lymph node of mice aged 4–5 months (n = 3 mice per genotype). Circles, cells from different mice. Lines, mean ± s.e.m. n, Flow cytometric analysis of mesenteric lymph node cells from the mice in m. Percentages represent the mean ± s.e.m. The three gates in l were also applied to these samples. For gel source data, see Supplementary Fig. 1.
