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. 2017 Apr 25;19(4):785–797. doi: 10.1016/j.celrep.2017.04.010

Figure 5.

Figure 5

Caspase-8 Scaffold Function Is Indispensable for DISC Recruitment of DED Proteins

Caspase-8a (C8a) and its respective active site mutant (C8a ASM) were reconstituted in caspase-8-deficient HeLa (C8 CRISPR) cells.

(A) Parental and HeLa C8 CRISPR cells either overexpressing the empty vector (CTRL), C8a, or C8a ASM were treated with 10 nM 4-hydroxytamoxifen (4-HT) for 6 hr to induce the expression of the respective constructs. Cells were pre-treated with 10 μM zVAD-fmk (zVAD) for 1 hr followed by stimulation with 5 U/mL CD95L-Fc for 3 hr. Cell viability was analyzed in triplicates by crystal violet staining. Shown are mean values ± SEM of three independent experiments. Significance levels (p values) were measured by two-way ANOVA test (∗∗∗∗p < 0.0001). Expression of C8a and C8a ASM were analyzed by western blotting.

(B) As described in (A), C8a was reconstituted in HeLa C8 CRISPR cells for 6 hr by 4-HT. Cell lines were stimulated with 2 U/mL CD95L-Fc for the indicated time points. CD95 was immunoprecipitated from cell lysates (TL) and analyzed for DISC-associated proteins. Asterisks mark non-specific bands.