Fig. 2.

eNOS expression influences the polarization state of endothelial cells. A Schematic representation of Golgi apparatus orientation measurements during a wound healing migration assay of confluent cell monolayers. Cells were subsequently fixed and stained for GM130 (Golgi) (red) and the nucleus (blue). Positions of the nucleus (blue) and the Golgi (red) are represented as a function of the position of the wound. The number of orientated cells was quantified in the first 2–3 layers and reported relative to the total number of cells in those layers. B Confluent CT-siRNA and eNOS-siRNA transfected BAECs were scratched and treated with VEGF as indicated for 30 min. The leading edge of cells at the migration front was made visible by F-actin staining (phalloidin, white). Representative images are shown. C Quantification of polarized transfected BAECs. D Mouse lung endothelial cells (MLECs) derived from eNOS^−/− mice and controls eNOS^+/+ were scratched and treated with VEGF for 30 min where indicated. E Quantification of polarized MLECs is shown. Results are displayed as mean values ± SEM. *p < 0.05. The graphs are representative of three independent experiments yielding similar results. F Quantification of polarized VEGF-stimulated BAECs that were transfected with CT-siRNA, eNOS-siRNA, Par3-siRNA or both Par3- and eNOS-siRNA. Depletion of eNOS and Par3 were monitored by immunoblot (IB). β-actin was used as a loading control. At least 50 cells and 25 cells per condition were quantified for BAECs and MLECs, respectively. *p < 0.05