Figure 3.

3TSR stimulates caspase-8, caspase-3, and cytochrome c release from mitochondria and promotes activation of caspase-9 in HDMEC. A, HDMEC cells were treated with buffer (CTL), 3TSR, hTSP-1, and caspase-8 (z-IEDT-fmk), caspase-3 (z-DEVD-fmk), or caspase-9 (z-LEHD-fmk) inhibitor. Caspase cleavage was detected by Western blotting with specific caspase-8. Caspase-3 or caspase-9 antibodies. Jurkat cells treated with TRAIL in the presence or absence of z-IETD-fmkwere used as controls. B, cytochrome c (Cyt C) levels in mitochondrial and cytosolic fractions were determined by Western blotting after treatment of cells with 2 or 5 μmol/L 3TSR or buffer. Jurkat cells treated with or without Fas ligand for 6 h were used as controls. Western blot analysis indicated that the mitochondrial preparations were free of GRB2, a protein that is normally excluded from mitochondria. C, HDMEC were treated with 3TSR in the absence or presence of the caspase-8 inhibitor z-IETD-fmk, the caspase-9 inhibitor z-LRHD-fmk, either alone or in combination, and the caspase-3 inhibitor z-DEVD-fmk. Camptothecin was used as a positive control for induction of apoptosis in endothelial cells. Mean ± SD of three separate experiments.