Caspase-10 in Fas Signalling Figure 3. Caspase-8 and -10 can restore FasL-induced apoptosis in caspase-8 and -10-doubly deficient Jurkat cells. A, B, I9-2e Jurkat cells were transiently transfected with plasmids encoding EGFP, EGFP-tagged wild-type caspase-8 or -10 or EGFP-tagged catalytically inactive caspase-8 mutant (Casp-8 C360S). Alternatively, cells were co-transfected with plasmids encoding EGFP and catalytically inactive caspase-10 mutant (C401S). Twelve hours post-transfection, cells were further incubated for 4 hours in the presence or absence of 500 ng/mL FasL. Cells were labelled with annexin-V-APC (AnV-APC). EGFP fluorescence was monitored on untreated cells to determine the percentage of transfected cells (A). Analysis of annexin-V binding was restricted to the EGFP-expressing cells on both untreated (None) and FasL-treated (FasL) cells (B). Percentages of fluorescent cells are indicated. Data are from a representative experiment out of three independent experiments. C, Specific cell death triggered by FasL was calculated by subtracting basal values obtained in the absence of FasL for each transfection. Data are means 6 SEM of three independent experiments. *p,0.05; **p,0.01. doi:10.1371/journal.pone.0013638.g003 of the dead cells were doubly-labelled by annexin-V and propidium iodide (Fig. 4C). Whereas I9-2d cells, which express caspase-10, died to a similar extent as A3 cells in the presence of zVAD-fmk, I9-2e cells remained completely resistant to FasL (Figs. 4B, C and D). Altogether, these results highlight the putative role of endogenous initiator caspase-10 in the form of cell death triggered by FasL in the presence of zVAD-fmk. Ectopic expression of wild-type caspase-10 restores FasL- induced cell death in zVAD-fmk-treated I9-2e cells To further evaluate the putative role of initiator caspases in FasL-induced cell death in the presence of zVAD-fmk, wild-type or catalytically inactive caspase-8 or -10 were expressed in I9-2e cells and, immediately after transfection, cells were incubated in the presence of 40 mM zVAD-fmk. Twelve hours post-transfec- tion, cells were further incubated for 4 hours in the presence or absence of FasL and analysed by flow cytometry (Fig. 5A). Whereas, in the presence of zVAD-fmk, no toxicity was induced by wild-type caspase-8 and caspase mutants, over-expression of wild-type caspase-10 slightly enhanced annexin-V labelling (Fig. 5B). Noteworthy was the finding that over-expression of caspase-10 restored FasL-induced annexin-V labelling in the presence of zVAD-fmk (Figs. 5B and C). Over-expression of catalytically inactive mutants failed to sensitize cells to FasL under our experimental conditions (Fig. 5C). Altogether, our data provide genetic evidence for the involvement of caspase-10 in FasL-induced cell death in the presence of zVAD-fmk, which was previously defined as ‘‘caspase-independent cell death’’. Moreover, caspase-10 activity appears to be critical for inducing this form of cell death in response to FasL. zVAD-fmk does not abrogate caspase-10-triggered apoptosis To further evaluate the role of caspase-10 in Fas signalling, we attempted to perform Western blot analysis of caspase activation in reconstituted I9-2e Jurkat cells. However, the too low transfection efficiency of Jurkat cells precluded such analyses. We therefore used another cell type, i.e., the HeLa carcinoma cell line, which does express endogenous caspase-8 but not caspase-10 as compared to A3 Jurkat cells (Fig. 6A). HeLa cells could be efficiently transfected, as illustrated by high percentages of EGFP- expressing cells sixteen hours post-transfection with a plasmid PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13638