AG-490 consistently induced annexin-V-FITC binding in leukemic LGLs but not sensitization of Fas. (a) The percent specific apoptosis was calculated for normal PBMCs (NL), leukemic LGLs (patient number), and U266 cells after treatment with DMSO. Open bars, anti-Fas (CH11); gray bars, 50 μM AG-490 (AG); filled bars, AG-490 with CH11. These results were representative of at least two experiments for each patient, and the assay was performed in duplicate. The only exception was patient 10129, for whom the procedure was done only once because therapy was soon initiated. AG-490–induced apoptosis in leukemic LGLs was statistically significant with P < 0.05 by ANOVA. (b) Three-color FACS analyses were performed on PBMCs from three patients with LGL leukemia. The patient’s cells were first stained with an isotype control antibody-PE or anti–CD8-PE before staining with annexin-V FITC and 7-AAD. A live gate was used to collect either the CD8⁺ cells or the CD8^– cells, and then apoptosis was assessed (c). AG-490 was added to leukemic LGLs (patient 10160), and U266 cells or CH11 was added to CEM after pretreatment with medium (filled bars), or the caspase-3 inhibitor Ac-DEVD-fmk at doses of 25 μM (dark gray bars), 50 μM (open bars), and 100 μM (light gray bars). The data are shown as percent specific apoptosis and were performed in duplicate.