Caspase-10 in Fas Signalling Figure 8. zVAD-fmk does not totally abrogate FasL-triggered apoptosis in HeLa cells expressing caspase-10 at low level. A, B, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 mM zVAD-fmk for 1 hour and further incubated for 16 hours with or without 1 mg/mL FasL as indicated. Cells were labelled with Syto13 and propidium iodide before fluorescence microscopy examination (A). Of note, less than 5% of the cells were stained by propidium iodide under all conditions indicating that FasL-induced necrosis was marginal in HeLa cells. Percentages of cell death (i.e., cells exhibiting nuclear fragmentation and/or condensation) were determined by analysing at least 500 cells for each condition. Values are means 6SEM of three independent experiments (n.s.: not significant; **p,0.01.) (B). C, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 mM zVAD-fmk for 1 hour and further incubated for 8 hours with or without 1 mg/mL FasL as indicated. Western blot analysis was performed using the indicated antibodies. (*: EGFP-tagged pro-caspase-10, **: EGFP-tagged small catalytic caspase-10 subunit). doi:10.1371/journal.pone.0013638.g008 nuclear shrinkage (see Fig. 4B), suggesting that the cells underwent apoptosis-like or necrosis-like cell death in response to FasL. Of particular interest was the finding that endogenous caspase-10 was likely involved in this FasL-induced alternative form of cell death. Indeed, FasL was unable to kill cells in the presence of zVAD-fmk in two different caspase-8 and -10-doubly deficient Jurkat clones (i.e., I9-2e and I9-2a) (see Fig. 4 and data not shown) as well as in HeLa cells, which did not express endogenous caspase-10 (see Fig. 8). In marked contrast, I9-2d (i.e., caspase-8-deficient but caspase-10-proficient) and wild-type A3 Jurkat cells were equally sensitive towards the combination of FasL and zVAD-fmk (see Fig. 4). Genetic correction of I9-2e cells with a plasmid encoding EGFP-tagged caspase-10, but not caspase-8, restored FasL- induced cell death in the presence of zVAD-fmk (see Fig. 5), further arguing for a role of caspase-10 in FasL-induced alternative form of cell death. Caspase-10 activity was required for its ability to mediate cell death since the expression of a catalytically inactive caspase-10 (C401S) had no cytotoxic effect. In addition, FasL-induced cell death was not totally abrogated by zVAD-fmk in HeLa cells stably expressing caspase-10 at low level (see Fig. 8). The broad-spectrum caspase inhibitor zVAD-fmk is commonly used to study the role of caspases in cell signalling. However, our findings show that zVAD-fmk is not potent enough to fully inhibit caspase-10-mediated cytotoxicity. Accordingly, caspase-10 pro- cessing in Fas signalling still occurred in the presence of zVAD- fmk (see Figs. 6 and 8). Therefore, in the presence of zVAD-fmk, the residual protease caspase-10 activity likely promotes an PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13638