Fig. 1. Cell viability, caspase-3 activation, DNA fragmentation and surface exposure of phosphatidylserine in FasL-stimulated A20 B-lymphoma cells. (A) Cells were incubated for 30 minutes without (-) or with either 100 µM Ac-DEVD-cho (DEVD or D), 100 µM Ac-YVAD-cho (YVAD or Y) or 10 µM zVAD-fmk (zVAD or z). Then, cells were incubated for another 16 hours either without (white bars) or in the presence of 100 ng/ml of FasL (gray bars), and cell viability was determined by the MTS assay. (B) Caspase-3 activity was determined using the substrate Ac-DEVD-amc after incubation of cells (0-8 hours) with 10 (open circles), 32 (filled circles) or 100 (open squares) ng/ml FasL. (C) Cells were treated as described in (A), and after 4 hours of incubation with FasL, DNA fragmentation was analyzed by electrophoresis in agarose gels. (D) A20 cells were incubated (0 to 5 hours) with 100 ng/ml of FasL alone or 3 hours with FasL following pre-incubation with the inhibitor Ac-DEVD-cho (D) or zVAD-fmk (z), and then phosphatidylserine exposure on the cell surface was detected using Annexin-V-FITC. Only PI-negative cells, which represented approximately 90% of the total cell population, are shown. Data shown are the means with standard deviations from (A,B,D) or results representative of (C) three independent experiments.