Involvement of PI3K/Akt-dependent NO production in RANKL-induced migration and capillary-like network by ECs. (A,E) HUVECs were preincubated for 30 minutes with or without 100 nM Wortmannin or 1 mM NMA prior to stimulation with RANKL (5 μg/mL). (C, F) HUVECs were stably transfected with DN-T2 and DN-T6 using retroviral system. (A,C) Cells were plated on Matrigel-coated plates at a density of 2 × 105 cells/well and incubated with 5 μg/mL RANKL. Microphotographs were taken after 20 hours (× 200). (B,D), Capillary-like networks were quantified with Image-Pro Plus software. (E-F) After 4 hours of incubation, chemotaxis was quantified with an optical microscopy. Three independent experiments were performed in duplicate. Data are means ± SDs; *P < .05; * *P < .01 versus RANKL alone.
