Fig. 5: CYLD depletion rescues defective M1-ubiquitylation in Complex I and consequently protects A20-deficient cells from TNF-induced apoptosis. | Cell Death & Disease

Fig. 5: CYLD depletion rescues defective M1-ubiquitylation in Complex I and consequently protects A20-deficient cells from TNF-induced apoptosis.

From: A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms

Fig. 5: CYLD depletion rescues defective M1-ubiquitylation in Complex I and consequently protects A20-deficient cells from TNF-induced apoptosis.The alternative text for this image may have been generated using AI.

a A20+/+ and A20−/− MEFs were transfected with siRNA targeting CYLD or non-specific siRNA (NS) and stimulated with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 Complex I was subsequently FLAG-immunoprecipitated. Protein levels were determined by immunoblotting. b–d A20−/− MEFs were transfected with siRNA targeting CYLD or nonspecific siRNA (NS). Cells were pretreated with the indicated compounds for 30 min before stimulation with 10 ng/ml mTNF. Cell death markers were monitored by immunoblotting (b) and cell death was measured in function of time by SytoxGreen (SG) positivity (c, d). e A20−/− HaCaTs were transfected with siRNA targeting CYLD or nonspecific siRNA (NS). Cells were pretreated with CHX for 30 min before stimulation with 10 ng/ml hTNF. Cell death was measured in function of time by SytoxGreen (SG) positivity. f, g A20+/+ Cyld+/+ (n = 3), A20ΔIEC Cyld+/+ (n = 3), and A20ΔIEC Cyld ΔIEC (n = 3) were injected i.p. with 5 µg/20 g mTNF. Caspase activity was quantified from small intestinal tissue homogenates 2 h post injection using a fluorescent caspase-activity probe (DEVD-AMC) (f) or via immunoblotting (g). h, i A20+/+ Cyld+/+ (n = 5), A20ΔIEC Cyld+/+ (n = 4), and A20ΔIEC Cyld ΔIEC (n = 5) were injected i.p. with 5 µg/20 g mTNF. Cumulative survival rates (h) and body temperature (i) were determined in function of time. Temperatures are presented as mean ± SEM. Statistical significance for body temperatures of the mice was determined using two-way ANOVA followed by a Tukey post hoc test. Survival curves were compared using log-rank Mantel–Cox test. Cell death experiments are presented as mean ± SEM of three independent experiments. Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey post hoc test. Significance between samples is indicated in the figure as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ns non significant.

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