Flow cytometric analysis of HUVECs showing increased levels of activated caspases. HUVECs were cotransfected with a transposon expressing DsRed 2 plasmid and shRNA specific to Beclin 1 or a scrambled shRNA, using Lipofectamine 2000. After 36 hours, transfected HUVECs were treated with 1.5 μg/mL rK5. Treated cells were labeled with green fluorescent-labeled inhibitor of caspases (FLICA) and analyzed by FACS. Transfected HUVECs were gated for DsRed⁺ cell populations and scored for FAM-VAD-FMK⁺ cells. (A) Histogram shows FAM-VAD-FMK⁺ cells in the control, untransfected, scrambled shRNA-transfected, and shRNA specific to Beclin 1–transfected HUVECs. (B) Histogram of rK5-treated HUVECs that were transfected with either shRNA specific to Beclin 1 or scrambled shRNA. (C) Summary of the data from the flow cytometric analyses is depicted as a percentage (mean ± SE) of transfected HUVECs (DsRed 2) positive for the activation of caspases. Each value is a mean of triplicate determinations.