Figure 3.

Involvement of caspase activity in endothelial cell apoptosis induced by shape restriction. (A) Representative experiment showing caspase-3 activity in suspended cells (sus) or in cells on fibronectin-coated dishes without (spread) or with exposure to Cyto D (1 μg/ml), Noc (10 μg/ml), BDM (10 mM), or Y27632 (Y27; 20 μg/ml) for 24 h; similar results were obtained in two or more experiments. Black bars, untreated cells; striped bars, cells pretreated with the caspase inhibitor z-VAD.fmk (100 μM) for 1 h and then continually exposed to the inhibitor during the 24-h experiment. (B) Prevention of apoptosis in shape-restricted cells treated with a caspase inhibitor. Capillary endothelial cells were grown on 10-μm circles (10 micron) or treated with Cyto D (1 μg/ml) for 24 h in the presence (striped bars) or absence (black bars) of the caspase inhibitor z-VAD.fmk (100 μM). Apoptosis was measured as the percentage of nuclei stained positively by the TUNEL enzyme reagent. (C) Caspase-8 activity in adherent cells treated in the absence (control) or presence of Cyto D (1 μg/ml), or in cells grown in suspension without (sus) or with the caspase inhibitor, z-VAD.fmk (100 μM), for 20 h. Caspase activities are expressed as the ratio of fluorescence units between treated and control cells.