Caspase-10 in Fas Signalling Figure 4. Impairment of FasL-induced cell death in the presence of zVAD-fmk in caspase-8 and -10-doubly deficient Jurkat cells. A, A3 cells were incubated for the indicated times with 500 ng/mL FasL in the presence (open symbols) or absence (filled symbols) of 40 mM zVAD-fmk. Caspase activities were assessed using Ac-DEVD-AMC (circles) or Ac-IETD-AMC (triangles). Data are representative of two independent experiments. B, A3 and I9-2e cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL and zVAD-fmk (40 mM) as indicated. Cells were fixed and stained with DAPI before microscopy examination. Arrows indicate cells with partial chromatin condensation. C, D, A3, I9-2d and I9-2e cells were incubated for 8 hours in the presence of zVAD-fmk (40 mM) with or without 500 ng/mL FasL as indicated. Cells were stained with Annexin-V-FITC (AnV-FITC) and propidium iodide (PI) and analysed by flow cytometry. Representative flow cytometry experiment out of four independent experiments (C). Low right quadrants: percentages of [AnV-FITC+ PI-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells. D, Percentages of anexin-V-positive (AnV+) cells (means 6 SEM, n = 4). **p,0.01. doi:10.1371/journal.pone.0013638.g004 encoding EGFP, EGFP-tagged caspase-8 or -10 (Fig. 6B), allowing caspase expression analysis by Western blot. To evaluate the caspase-inhibitory efficacy of zVAD-fmk under our experimental conditions, HeLa cells were transiently transfected with the different constructs and immediately treated with 40 mM zVAD- fmk and further incubated for 24 hours. During the last 8 hours, 1 mg/mL FasL was added or not to the cell culture medium. In cells transfected with plasmids encoding either EGFP-tagged caspase-8 or -10, an approximately 40 kDa band was revealed with anti-EGFP antibody (Fig. 6C). The appearance of this band did not require FasL treatment and most likely corresponded to the EGFP-tagged small catalytic subunit of pro-caspase-8 and -10, indicating auto-processing of the over-expressed enzymes (Fig. 6C). The addition of zVAD-fmk led to a different pattern depending on the type of EGFP-tagged initiator caspase expressed. Indeed, the 40 kDa band decreased and accumulated in EGFP-tagged caspase-8 and -10 expressing cells, respectively. In the meantime, whereas EGFP-tagged pro-caspase-8 strongly accumulated, pro- caspase-10 accumulation was less pronounced (Figure 6C). The consequences on endogenous caspase-9 and PARP cleavage were next analysed under our different experimental conditions. In the absence of zVAD-fmk, caspase-9 processing (i.e., disappearance of pro-caspase-9) occurred upon FasL treatment, this phenomenon being slightly enhanced in EGFP-tagged expressing caspase-8 or -10 cells. The addition of zVAD-fmk fully prevented caspase-9 and PARP cleavage in EGFP and EGFP- PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13638