Caspase-10 in Fas Signalling Figure 5. Caspase-10 can restore FasL-induced cell death in the presence of zVAD-fmk in caspase-8 and -10-doubly deficient Jurkat cells. A, B, I9-2e Jurkat cells were transiently transfected as described in the legend to Figure 3 and immediately incubated in the presence of 40 mM zVAD-fmk. 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). EGFP fluorescence was monitored on untreated cells to determine the percentage of transfected cells (A). Annexin-V binding analysis 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. Of note, the present data were obtained in the same experiment depicted in Fig. 3B. 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,0.01. doi:10.1371/journal.pone.0013638.g005 tagged caspase-8 expressing cells. Of particular interest was the finding that caspase-9 and PARP processing was not totally abrogated in EGFP-tagged caspase-10 expressing cells (Fig. 6C). Accordingly, whereas zVAD-fmk completely prevented caspase-8- triggered toxicity, as evaluated by annexin-V labelling, cell death still occurred in caspase-10 over-expressing cells (Fig. 6D). Collectively, our data indicate that, whereas zVAD-fmk potently prevented caspase-8 auto-processing and pro-apoptotic activity, it had a partial effect on caspase-10 pro-apoptotic signalling in an over-expression system. zVAD-fmk does not inhibit caspase-10 processing in Fas signalling We then attempted to stably express caspase-10 in HeLa cells at a physiological level to further evaluate its role in Fas signalling. HeLa cells transfected with a plasmid encoding EGFP-tagged wild-type caspase-10 were cultured under G418 for 45 days. G418-resistant HeLa cells were analysed by flow cytometry and less than 10% of cells expressed EGFP-tagged caspase-10 at low levels (Fig. 7A). Indeed, expression was 3 to 4 times lower in stably than in transiently transfected cells (compare Figs. 6B and 7A). From this heterogeneous cell population, two cell populations were FACS sorted based on EGFP expression: EGFP-tagged caspase-10 expressing (Casp10+) HeLa cells and their control counterparts (Casp10-), which did not express EGFP at all (Fig. 7B). Those were further grown under G418. This protocol allowed the establish- ment of Casp10+ HeLa cells, among which 60% of cells stably expressed EGFP-tagged caspase-10 as evaluated by flow cytometry (Fig. 7C). Casp10+ and Casp10- HeLa cells displayed similar CD95, FADD, RIP and caspase-8, -9 and -3 expression as evaluated by Western blot and flow cytometry (Figs. 7C and D). Whereas Casp10+ and Casp10- HeLa cells did not express endogenous caspase-10, Casp10+ HeLa cells expressed exogenous EGFP-tagged caspase-10 at low level as compared to endogenous caspase-10 expression in A3 Jurkat cells (Fig. 7D). Casp10+ HeLa cells and their control counterparts (Casp10-) were pre-incubated with or without 40 mM zVAD-fmk for one hour and further incubated in the presence or absence of FasL. Cell morphology analysis indicated that FasL induced apoptosis (i.e., nuclear condensation and/or fragmentation) in both cell lines, caspase-10-proficient cells being slightly, yet not significantly, more sensitive than caspase-10-null cells (Figs. 8A and B). Addition of zVAD-fmk fully prevented FasL-triggered cell death in caspase- 10-null HeLa cells. In sharp contrast, apoptotic cell death was not PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13638