alternative route of cell death, leading to apoptosis-like cell death. Thus, experiments based on the use of zVAD-fmk to study the caspase-independent pathway should be interpreted with caution. Moreover, our study challenges the existence of an alternative caspase-independent cell death pathway in Fas signalling. The previously so-called ‘‘caspase-independent cell death’’ triggered by death receptors has been reported to be RIP-dependent [15]. One should note that FasL-induced caspase-dependent apoptosis was slightly, yet significantly, reduced in RIP-deficient Jurkat cells [25]. Thus, we cannot rule out the possibility that RIP may functionally interact with caspase-10 to mediate the activation of an alternative cell death. This hypothesis is strengthened by the observation that FADD was required for FasL-induced cell death in the presence of zVAD-fmk [15]. Indeed, whereas FADD deficiency in Jurkat cells did not impair RIP recruitment to Fas/CD95 in response to FasL, FADD-deficient Jurkat cells were fully resistant to both FasL-induced apoptosis and ‘‘caspase-independent cell death’’ [15]. Different groups have previously reported that FADD, besides its capacity to activate the classical caspase cascade activation, may also trigger a non-apoptotic form of cell death [26,27,28,29,30]. Interestingly, this non-apoptotic form of cell death occurred in normal but not in prostate cancer epithelial cells, indicating that this alternative pathway initiated by FADD may be disrupted in some cancer cells [29]. Mutations affecting Fas, FasL, caspase-8 or -10 may be responsible for ALPS or malignancies [2,31,32]. It is tempting to speculate that some mutations may affect the caspase-10- dependent cell death pathway, thereby contributing to FasL resistance in ALPS or in malignant diseases. Materials and Methods Reagents Final concentrations or dilutions of the following reagents are indicated: zVAD(OMe)-fmk (40 mM) was from Bachem (Voisins- Le-Bretonneux, France); monoclonal anti-FADD (clone A66-2; 0.5 mg/mL), anti-RIP (clone G322-2, 0.25 mg/mL) and anti- caspase-8 (clone B9-2; 0.5 mg/mL) antibodies were from BD Biosciences (Le Pont-de-Claix, France); monoclonal anti-caspase- 10 (clone 4C1; 1 mg/mL) was from MBL (Meudon, France); polyclonal anti-caspase-3 (10 mg/mL) was from Dako (Trappes, France); monoclonal anti-caspase-2 (clone C2) and polyclonal anti- caspase-7, anti-caspase-9, anti-PARP and anti-Bid antibodies were from Cell Signaling Technology (Saint-Quentin-en-Yvelines, France) and used at 1/1000 dilution; monoclonal anti-b-actin (clone AC-15; 5 mg/mL) was from Sigma (Saint-Quentin-Falla- vier, France); monoclonal anti-CD95 (clone 7C11; 1/5 dilution) and IgM irrelevant antibody (10 mg/mL) coupled to phycoerythrin (PE) were purchased from Immunotech (Marseille, France) and Santa Cruz (Le-Perray-en-Yvelines, France), respectively. cDNAs encoding wild-type and catalytically inactive mutant caspase-8 (C360S) and wild-type caspase-10 [21] were subcloned into pEGFP.N1 plasmids. Catalytically inactive mutant caspase-10 (C401S) cDNA [21] was subcloned into pCiNeo plasmid. Plasmids were kindly given by Dr. M. Lenardo (Bethesda, MD). Mouse FasL produced in the culture medium of Neuro-2A cells stably transfected with a plasmid encoding FasL was used at a final concentration of 500 ng/mL [13,33]. Alternatively, human FasL kindly provided by Dr. P. Legembre (EA 4427 SeRAIC, Rennes University, France) was used at a final concentration of 1 mg/mL. Cell lines Parental Jurkat T leukaemia cells (clone A3) and the derived cell line deficient for caspase-8 (clone I9-2) were kindly provided by Caspase-10 in Fas Signalling Dr. J. Blenis (Boston, MA) [23]. I9-2a, b, d and e cells were isolated by limiting dilution experiments from I9-2 cells as previously described [13]. Cells were cultured in RPMI 1640 medium containing Glutamax and 10% heat-inactivated fetal calf serum (FCS). HeLa cells were from ATCC and cultured in DMEM medium containing Glutamax and 10% FCS. Flow cytometry analyses For studying phosphatidylserine externalization, cells were labelled with annexin V-FITC (250 ng/mL) and propidium iodide (12.5 mg/mL) (Immunotech, Marseille, France) for 10 min at 4uC. Alternatively, cell death of EGFP fluorescent cells was monitored after labelling with annexin V-APC (250 ng/mL) (Immunotech, Marseille, France) for 10 min at 4uC. CD95 cell surface expression was determined after incubation of cells for 30 min at 4uC with or without anti-CD95-PE or an irrelevant antibody coupled to PE. Analyses were performed on a FACScan (Becton Dickinson, Le- Pont-de-Claix, France) cytometer. MTT assay Viability was evaluated by the tetrazolium-based MTT assay. Cells were seeded in flat-bottom 96-well plates (106 cells/mL, 100 mL/well) for 8 hours at 37uC. After 2-hour incubation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma), formazan crystals were solubilized overnight at 37uC by adding 100 mL of solubilization buffer (HCl 0.01 N, 10% SDS) and spectrophotometrically quantified (l = 590 nm). Protein extraction, Western blotting analyses and fluorogenic DEVD and IETD cleavage enzyme assays For total protein extraction, 5 to 206106 cells were lysed for 30 min on ice in a buffer containing 10 mM HEPES (pH 7.4), 42 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5% CHAPS, 1 mM PMSF and 2 mg/mL leupeptin. Samples were centrifuged at 10,000 g at 4uC for 10 min. Supernatants were collected and protein content was determined by the Bradford method (Biorad). For Western blot analyses, equal amounts of protein were separated on 15% SDS-PAGE. Caspase activities were assessed using Ac-DEVD-AMC or Ac-IETD-AMC substrates (Bachem) as described elsewhere [34]. Morphological analyses 100,000 Jurkat cells were sedimented onto microscope slides by cytospin at 100 g for 10 min. Cells were fixed by 0.4% paraformaldehyde, DAPI stained and examined under a fluores- cence Leica microscope. Alternatively, HeLa cells were labelled with 2.5 mM Syto 13 (Molecular probes, Leiden, The Netherlands) and 2 mg/mL propidium iodide (Sigma). The percentage of apoptotic cells (having nuclear condensation and/or fragmenta- tion) was evaluated by counting at least 500 cells for each condition under a Leica fluorescence-equipped inverted micro- scope as previously described [13]. Transfection experiments Jurkat cells were transiently transfected as previously described [35]. Briefly, 26106 cells were incubated in 0.2 mL 10 mM phosphate buffer (pH 7.4) containing 250 mM sucrose and 1 mM MgCl2. Eight mg of plasmids encoding EGFP, EGFP-tagged wild- type caspase-8, EGFP-tagged C360S caspase-8 or EGFP-tagged wild-type caspase-10 were added before electro-pulsation. Alter- natively, cells were co-transfected by 2 mg of the plasmid encoding EGFP and 8 mg of the plasmid encoding C401S caspase-10. PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13638