Disruption of Survivin ΔEx3 inhibits vascular tube formation in vitro. (A) Schematic representation of the Survivin gene structure and shRNA targeting regions. Colored boxes represent exons from the SURVIVIN gene. The 5 identified splice variants are represented, and the targeting region is shown as an asterisk above the area targeted. The arrow represents the location of the translational stop in each of the mRNAs. Numbers 1 to 3 consist of the combination of shRNAs previously described,¹²  whereas number 4 represents the Survivin ΔEx3–specific shRNA. (B) HUVE cells were treated with VEGFR1 or VEGFR2 blocking antibodies (50 ng/mL) or supplemented with VEGF (20 ng/mL); (C) HUVECs were transfected with scrambled shRNA, non–Survivin ΔEx3 shRNA (SH3-9), Survivin ΔEx3 shRNA, or a combination of 3 Survivin shRNAs; or (D) HUVECs were treated with Survivin ΔEx3 antibody (10 μg/mL), Survivin ΔEx3 antibody (10 μg/mL) plus VEGF (20 ng/mL), isotype control antibody (10 μg/mL), or actinomycin-D (7.5 μg/mL) and plated on growth factor–reduced BME in 96-well plates. For all conditions, tube formation was assayed at 4 and 16 hours and quantitative data were collected at the 16-hour time point by measuring the length of the tubules formed under an inverted light microscope at ×100 magnification (40×/0.75 objective lens). Quantitative data shown in panel E are mean ± SE of 5 replicates per sample.