Disruption of Survivin ΔEx3 does not induce apoptosis or affect proliferation in HUVE cells. HUVE cells were cotransfected with scrambled shRNA, Survivin ΔEx3 shRNA, or combination shRNA along with pHcRed. Red fluorescent cells were separated by FACS at 48 hours. (A) Cells were assayed for apoptosis by staining with annexin V. (B) Cells were grown in a 96-well plate and assayed for active caspase 3 with caspase 3/7 GLO. The y-axis represents the percentage of caspase 3 activity, relative to control. (C) Cells were pulsed with 10 μM BrdU for 1 hour, and staining was performed as described in “Materials and methods,” using an anti–BrdU-FITC or an IgG isotype-FITC antibody. Both apoptosis and proliferation were analyzed by flow cytometry. Experiments were performed in triplicate, and data are shown for a representative sample set. Error bars are the standard error of the mean of three replicates per sample.