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

Figure 6.

Figure 6

Caspase-10 Promotes CD95L-Mediated Gene Induction

(A and B) HeLa cells expressing shC10 or shCTRL were treated for 72 hr with 0.5 μg/mL doxycycline. (A) Duplicate wells were stimulated in media containing 0.5% FCS with the indicated concentrations of CD95L-Fc for 24 hr. Supernatants were analyzed for secreted interleukin-8 (IL-8) by ELISA. Cell viability was assayed using crystal violet staining. (B) HeLa ± shC10 cells were pre-starved for 4 hr in media containing 0.5% FCS followed by treatment with 10 μM zVAD-fmk (zVAD) for 1 hr. Cells were stimulated with 0.1 U/mL CD95L-Fc for 3 hr. RNA was isolated, reverse transcribed to cDNA, and mRNA expression levels of IL-8, IL-6, CCL2, EGR1, CXCL3, and TNF were analyzed by real-time qPCR.

(C) Parental and caspase-10-deficient (C10 CRISPR) HeLa cells were starved and pre-treated with zVAD as described in (B). Cells were stimulated with CD95L-Fc (0.0025, 0.005, 0.01, 0.025, 0.05, or 0.1 U/mL) for 3 hr. IκBα phosphorylation as well as degradation and caspase-10 knockout were analyzed by western blotting. Asterisks mark non-specific bands.

(D) Parental and caspase-8-deficient (C8 CRISPR) HeLa cells were treated with 10 nM 4-HT for 6 hr in media containing 0.5% FCS to induce the expression of either control plasmid or caspase-8a (expression of caspase-8a ASM was achieved in the absence of induction via 4-HT). Cells were stimulated with zVAD and CD95L-Fc as described in (B) and analyzed for IL-8 mRNA expression by real-time qPCR. Caspase-8 expression was quantified after reconstitution and compared to parental HeLa cells as indicated in the western blots. Relative IL-8 mRNA induction has been calculated with respect to caspase-8 expression.

(E) Parental, C10 CRISPR, and reconstituted caspase-10a/ASM HeLa cells were treated as described in (D) (expression of wild-type caspase-10 was achieved in the absence of induction via 4-HT). Cells were stimulated with zVAD and CD95L-Fc as described in (B) and IL-8 mRNA expression analyzed. Relative caspase-10 expression and IL-8 induction has been calculated as described in (D) (raw data for D and E are shown in Figures S6C and S6D).

Each graph/diagram represents mean values ± SEM of three independent experiments. Significance levels (p values) were measured by Student's t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).