caspase substrate Ac-IETD-AMC (Fig. 2A), and apoptosis, as evaluated by flow cytometry (Figs. 2B and C), in A3 cells and, albeit to a lesser extent, in I9-2d cells. FasL totally failed to trigger caspase activation and apoptosis in I9-2e cells (Fig. 2). Identical data have been found using the other caspase-8 and -10-doubly deficient cells (i.e., I9-2a). These data are consistent with our previous study indicating the involvement of both endogenous caspase-8 and -10 in FasL-induced apoptosis [13]. Over-expression of caspase-8 or -10 restores FasL- induced apoptosis in I9-2e cells Experiments were next restricted to I9-2e cells, which do not express endogenous caspase-8 and -10 (see Fig. 1A and [13]). Over-expression of EGFP, an irrelevant protein in cell death signalling, was used as a negative control to evaluate the basal cytotoxicity induced in our experimental settings. Cells were transfected with plasmids encoding EGFP-tagged wild-type or catalytically inactive caspase-8 (C360S). Alternatively, cells were transfected with a plasmid encoding EGFP-tagged wild-type caspase-10 or co-transfected with plasmids encoding EGFP and untagged catalytically inactive caspase-10 (C401S). Twelve hours post-transfection, cells were further incubated for 4 hours in the presence or absence of FasL (Fig. 3A). Cells were next labelled with annexin-V-APC and analyses were restricted to the EGFP- positive (i.e., transfected) cells. At least 1,000 EGFP-positive cells Caspase-10 in Fas Signalling were analyzed. As compared to EGFP alone expression, ectopic expression of either wild-type caspase-8 or -10 strongly enhanced annexin-V labelling (Fig. 3B), which was further increased upon FasL treatment (Figs. 3B and C). Annexin-V labelling was slightly increased when cells expressed the catalytically inactive caspase mutants (Fig. 3B). However, mutants failed to significantly sensitize cells to FasL (Fig. 3C). Altogether, our data further indicate that both initiator caspase-8 and -10 are involved in FasL- induced apoptosis. FasL-induced cell death in the presence of zVAD-fmk is impaired in Jurkat cells doubly deficient for caspase-8 and -10 In wild-type Jurkat cells, FasL-triggered caspase activation was fully abrogated by the addition of 40 mM zVAD-fmk to the cell culture medium (Fig. 4A). However, cell death, as evaluated both by microscopy (Fig. 4B) and flow cytometry (Fig. 4C) analyses, still occurred in the presence of zVAD-fmk. Microscopic analysis after DAPI staining indicates that, in the absence of zVAD-fmk, FasL elicited apoptosis in A3 cells as illustrated by strong nuclear condensation and/or fragmentation (Fig. 4B). In the presence of zVAD-fmk, dying cells displayed distinct nuclear features such as partial chromatin condensation in the absence of nuclear shrinkage, suggesting that FasL-triggered an alternative form of cell death (Fig. 4B). Flow cytometry experiments indicate that most Figure 2. Impairment of FasL-induced caspase activation and apoptosis in caspase-8 and -10-doubly deficient Jurkat cells. A, B, A3 (white bars), I9-2d (grey bars) and I9-2e (black bars) Jurkat cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL as indicated. Caspase activities were assessed using Ac-DEVD-AMC or Ac-IETD-AMC (A). Cells were stained with annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Percentages of annexin-V-positive (AnV+) are indicated (B). All data are means 6 SEM of three to four independent experiments. *p,0.05; **p,0.01, *** p,0.001. C, Representative flow cytometry experiment. Low right quadrants: percentages of [AnV-FITC+ propidium iodide (PI)-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells. doi:10.1371/journal.pone.0013638.g002 PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13638