mTOR Modulation Impacts Endothelial Angiogenesis

Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen (Burlington, ON). VEGF-A was from Peprotech (Princeton, NJ). Rapamycin, PP242, a highly specific mTOR active-site inhibitor [12 (link)], and anti-tubulin-α was from Millipore (Temecula, CA). Ku-0063794, a second specific mTOR inhibitor [13 (link)] was from Sigma (St. Louis, MO). Anti-Akt1 was from Protein Tech (Chicago, IL). Hiperfect, non-silencing short interfering RNA (siRNA) and Akt1 silencing siRNA were from Qiagen Inc (Mississauga, ON). Human tumor necrosis factor-α (TNFα) was from Cedarlane (Mississauga, ON). Cycloheximide, phalloidin-FITC, anti-vinculin, and DAPI were from Sigma. Anti-S6K was from Abcam (Cambridge, UK). Anti-phospho-Akt^S473, anti-phospho-S6K^T389, anti-FAK, anti-phospho-FAK^Y397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology (Danvers, MA). ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific (Waltham, MA).