Caspase-10 in Fas Signalling Figure 7. Generation of HeLa cells stably expressing EGFP-tagged wild-type caspase-10 at low level. A, HeLa cells were transfected with plasmid encoding EGFP-tagged wild-type caspase-10 and incubated for 45 days in DMEM 10% FCS containing 0.8 mg/mL G418. The whole population of cells (Casp10) were analysed by flow cytometry and untransfected HeLa WT cells were used as negative control to determine the threshold for FL1 channel. Values are percentages of positive cells in FL1 channel. B, Scheme for selection of cells expressing (Casp10+) or not (Casp10-) EGFP-tagged caspase-10. C, Casp10+ and Casp10- cells were FACS sorted from the heterogeneous population stably expressing caspase-10 and further cultured for one month in DMEM 10% FCS containing 0.8 mg/mL G418 before analysis by flow cytometry for EGFP-tagged caspase-10 and CD95 expression. Values indicated percentages of positive cells in FL1 (up panels) and FL2 (low panels) channels. D, Wild-type Jurkat (A3), Casp10+ and Casp10- HeLa cells were analysed by Western blot using the indicated antibodies. Caspase-10 expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-10). doi:10.1371/journal.pone.0013638.g007 Jurkat and HeLa cells expressing caspase-10. Thus, caspase-10 may participate, in addition to the classical pro-apoptotic pathway, to a zVAD-fmk-resistant alternative cytotoxic pathway mediated by FasL. Wild-type A3 Jurkat cells were highly sensitive to FasL (see Fig. 1C), and dying cells displayed classical features of apoptosis, including increased specific caspase activity (see Figs. 2A and 4A), phosphatidylserine externalisation (see Fig. 2A) and nuclear fragmentation (see Fig. 4B). Whereas caspase-8 and -10-doubly deficient I9-2e cells were fully resistant to FasL-induced caspase activation and apoptosis, caspase-8-deficient I9-2d cells, which expressed caspase-10, displayed an intermediate sensitivity as compared to I9-2e and A3 cells (see Figs. 1C, 2B and C). This was associated with a significant increase in specific caspase activity in I9-2d cells upon treatment by FasL (see Fig. 2A). These data provide genetic evidence that both endogenous initiator caspase-8 and -10 are involved in FasL-induced caspase-dependent cell death. Moreover, genetic correction of I9-2e cells either with EGFP-tagged wild-type caspase-8 or -10 restored FasL-induced annexin-V labelling, indicating that the resistance of I9-2e cells was unlikely a ‘‘clone effect’’ and further arguing that both initiator caspases are indeed involved in Fas apoptotic signalling. Over-expression of catalytically inactive caspase-8 and -10 in I9-2e cells slightly increased annexin-V labelling but failed to signifi- cantly sensitize cells to FasL (see Fig. 3). These findings are in accordance with recent observations indicating that, under some experimental conditions, caspase-8 may induce some cytotoxicity independently of its classical caspase activity [24]. During the last decade, FasL has been reported to trigger a RIP- dependent alternative form of cell death, exhibiting necrotic rather than apoptotic features [15]. This alternative form of cell death was initially defined as a caspase-independent cell death since it occurred in caspase-8-deficient Jurkat cells and was not sensitive to the broad-spectrum caspase inhibitor zVAD-fmk [15]. In accordance with this concept, FasL still triggered some cytotoxicity in A3 cells in the presence of zVAD-fmk (see Fig. 4D). Most of the dying cells were doubly labelled with annexin-V and propidium iodide (see Fig. 4C) and exhibited chromatin alterations with no PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13638