Fig. 5. Analysis of mini-Plg-induced apoptosis in ECs. (A) HUVECs cultivated in 20% serum medium in 96-well plates (5000 cells per well) were treated with 240 nM Plg or Plg fragments for 24 hours. After incubation for 24 hours, the cells were washed with cultivation medium and lysed. Intact DNA was removed by centrifugation and the presence of soluble nucleosomes in the supernatant was detected by the Death ELISA kit (Roche). The relative apoptotic response was determined using a standard included in the kit. HUVECs were treated with mini-Plg with or without the serine-protease inhibitor aprotinin (Apro, 10 µg ml^-1), the caspase-3 inhibitor DEVD-FMK (1 µM) or a neutralizing anti-TGF-ß antibody (10 µg ml^-1) for 24 hours. (B) Cells were treated for 24 hours with 240 nM Plg or mini-Plg in medium containing 1% serum. After incubation, the detached cells were pooled with the trypsinized adherent cells and assayed for the occurrence of apoptosis by FACS analysis using an apoptosis detection kit (Calbiochem, La Jolla, CA, USA). Viable cells were annexin-V negative and PI negative; nonviable necrotic cells or late apoptotic were annexin-V positive and PI positive; early apoptotic cells were annexin-V positive and PI negative. Similar results were obtained in three different experiments.