Angiogenin

Freshly isolated HUVEC were maintained in endothelial cell growth medium (EGM) supplemented with SingleQuots (EGM BulletKit CC-3124, Lonza). They were then plated in a 96-well plates (14 × 10³ cells/well) previously coated with growth factor-depleted Matrigel (Becton-Dickinson, Bedford, MA) in M199 medium supplemented with 20% FBS, 1% glutamine, 1% antibiotic–antimycotic solution (Biological Industries) and 50 U/100 ml heparin (Biomedical Technologies Inc., Stoughton, MA). Cells were treated with trT2-50m (2 μM), peptides or PBS, in addition to angiogenin (R&D Systems, Inc. Minneapolis, MN), or VEGF (Protein Laboratories, Rehovot, Israel) (1 μg/ml each). After 8 h of incubation at 37°C, the plates were photographed and the extent of tube formation was counted using Image J (NIH, Bethesda, MD) software. Five individual determinations were performed for each treatment. Statistical analysis was performed using one way ANOVA for multiple comparison and 2-sample t-test.

HUVEC (CC-2519) were purchased from LONZA (Walkersville, MD USA). HUVEC were maintained in M199 medium supplemented with 20% FCS, 1% glutamine, 1% antibiotic-antimycotic solution, and 50 U/100 ml heparin [19 (link)]. The cells were then plated at a density of 14,000 cells/well in a 96-well plate previously coated with growth factor-depleted Matrigel™ (Becton-Dickinson, Bedford, MA) in M199 medium containing 5% FCS and supplemented with angiogenin (R&D Systems Inc., Minneapolis, MN, USA), basic fibroblast growth factor (bFGF, PeproTech Inc., Rocky Hill, NJ, USA) or vascular endothelial growth factor (VEGF, PLR, Rehovot, Israel) at a final concentration of 1 μg/ml each. hrRNASET2 (2 μM final concentration), or Avastin was also added. After overnight incubation at 37°C, the plates were photographed and the extent of tube formation was assessed. Five individual determinations were performed for each treatment.