Caspase activity. Effect of RGDS treatment on caspase activity. (A) HUVECs seeded on collagen IV (50 μg/mL) were treated with RGDS (500 μg/mL) for 4 hours at 37°C. Caspase 3/7 enzymatic activity was evaluated by measuring the fluorescence of the Z-DEVD-R110 substrate. Caspase 1, caspase 8, and caspase 9 activity were quantified by the release of AFC from the substrates (DEVD-AFC, AEVD-AFC, and IETD-AFC, respectively). RGDS significantly activated caspase 8 (Cas-8; ^*P < .01) and caspase 9 (Cas-9; ^*P < .05), whereas it had no effect on caspase 3/7 (Cas-3/7) and caspase 1 (Cas-1) activation. Results represent the average ± SD of 3 experiments performed in duplicate. (B) Caspase 3/7 enzymatic activity was measured as reported for panel A. RGDS increased caspase 3 activity after 24 hours (P < .05). These experiments were carried out in triplicate. (C) Cells were incubated at 37°C with RGDS for 4, 16, and 24 hours. Caspase 3 activation was followed as function of pro-caspase cleavage by Western blotting, incubating with a polyclonal rabbit antihuman caspase 3 antibody. RGDS reduced the inactive form of caspase 3 (pro-caspase 3 MW 32 kDa) after 24 hours of treatment (^*P < .01), whereas at 4 hours and 16 hours it was ineffective. (D) Pro-caspases 8 and 9 (55 kDa and 47 kDa, respectively) were cleaved to the active forms (20 kDa and 37 kDa, respectively) after 4 hours of treatment, whereas the 20-kDa active subunit of caspase 3 was generated after 24 hours of treatment; the increase of the cleaved active forms was always paralleled by a corresponding decrease of the pro-caspase inactive forms. The equal loading was confirmed by Ponceau S solution staining. The figure refers to one representative experiment, whereas the quantification refers to 4 experiments.