Mis-regulation of centrosome duplication does not lead to apoptosis in endothelial cells. (A-H) HUVECs were stained with antiactivated caspase 3 (red), pericentrin (green), and DRAQ5 nuclear dye (blue) after treatment with high VEGF for 96 hours, with (A-D) or without (E-H) the apoptosis-promoting drug HNE for the final 48 hours. (F) Inset: Centrosomes at higher magnification. HNE-treated cells were positive for activated caspase 3, but high VEGF-treated HUVECs containing excess centrosomes did not stain for activated caspase 3. (I-L) HUVECs were stained with antipericentrin (green), anti-α-tubulin (red), and DRAQ5 to visualize mitotic figures. Bipolar spindles containing 2 centrosomes (I) or more than 2 centrosomes (J) were observed in addition to multipolar spindles (K-L) containing more than 2 centrosomes. (M) HUVECs were incubated with low or high VEGF for the indicated times, then fixed and stained for γ-tubulin and DRAQ5 for centrosome counts (96-hour low VEGF, n = 890; 240-hour low VEGF, n = 976; 96-hour high VEGF, n = 1023; 240-hour high VEGF, n = 897). Experiments were performed in triplicate. Scale bar represents 5 μm. **P < .001 vs 96-hour low VEGF. ***P < .0001 vs 96-hour low VEGF. #P < .05 vs 96-hour high VEGF. (A-L) Panels imaged with Zeiss LSM 5 Pascal and 63×/1.4 NA oil objective; samples were in Aqua/Polymount; images acquired with PASCAL Release Version 4.2 SP1 software and managed in Abobe Photoshop CS 2 9.0.