Caspase-10 in Fas Signalling manner [19]. Moreover, a novel caspase-10 isoform lacking the large and small protease subunits, has been recently reported to interact with RIP, activate NF-kB and induce cell death in the absence of PARP [poly(ADP-Ribose)polymerase] cleavage [20]. Whereas the involvement of caspase-8 in FasL-triggered apoptosis is well established, that of caspase-10 still remains a matter of debate. Indeed, overexpression of caspase-10 comple- mented caspase-8 deficiency in FasL-treated Jurkat cells in two independent studies [9,21], but not in another [22]. The latter study concluded that caspase-10 is indeed recruited to the DISC in response to TRAIL or FasL but cannot functionally substitute caspase-8 [22]. The present study was undertaken to further evaluate the function of initiator caspase-10 in FasL-induced cell death. Our data demonstrate that (i) FasL-induced cell death is abrogated in caspase-8- and -10-doubly deficient Jurkat cells (I9-2e clone), but not in caspase-8-deficient Jurkat cells expressing caspase-10 (I9-2d clone); (ii) expression of wild-type caspase-10 can restore the ability of FasL to trigger cell death in I9-2e cells; (iii) zVAD-fmk does not impede FasL-induced caspase-10 processing and cytotoxicity. Results FasL-induced apoptosis is impaired in Jurkat cells doubly-deficient for caspase-8 and -10 To characterize the Jurkat variants, I9-2a, b, d and e derived from I9-2 cells, the expression of proteins involved in Fas signalling was investigated. As expected from previous observations [13,23], I9-2a, b, d and e cells did not express caspase-8 in contrast to A3 cells. However, FADD, RIP, caspase-2, -9, -3 and -7 (Fig. 1A) and Fas/CD95 (Fig. 1B) were equally expressed in A3 and in the I9-2 variants. Importantly, whereas caspase-10 was expressed in A3, I9- 2d cells and, albeit to a lesser extent, I9-2b cells, barely detectable expression of caspase-10 was found in I9-2a and e cells (Fig. 1A). When the different cell variants were incubated in the presence of FasL, toxicity was observed in A3 cells and, albeit to a lesser extent, in I9-2b and I9-2d cells (Fig. 1C). In sharp contrast, FasL- induced cell death was totally abrogated in the caspase-8 and -10- doubly deficient I9-2a and e cells (Fig. 1C). Accordingly, FasL induced caspase activation, as evaluated by the cleavage of the effector caspase substrate Ac-DEVD-AMC and the initiator Figure 1. Impairment of FasL-induced cell death in caspase-8 and -10-doubly deficient Jurkat cells. A, 20 mg total protein extracts from parental (A3) and caspase-8 deficient (I9-2a, b, d and e) Jurkat cell lines were subjected to SDS-PAGE, and Western blotted with anti-caspase-8, -10, -2, -9, -3, -7, anti-FADD, anti-RIP or anti-b-actin antibodies. B, Jurkat cells were labelled with anti-CD95-PE antibody (thick line) or an isotype control (thin line) and analysed by flow cytometry. Percentages of cells expressing CD95 are indicated. C, Jurkat cells were incubated for 8 hours in the presence of 500 ng/mL FasL and cell viability was evaluated by MTT assay. Values are means 6 SEM of three independent experiments. *p,0.05; **p,0.01, *** p,0.001 as compared to values obtained in A3 cells. doi:10.1371/journal.pone.0013638.g001 PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13638