Ang2 bdwt

Serum-reduced Matrigel (10 mg/ml; BD Biosciences) was thawed overnight at 4 °C, and 150 μl was added to each well of a 48-well microtiter plate and allowed to solidify for 1 h at 37 °C. The wells were incubated with 3.25 × 10⁴ TIME cells plus 500 ng/ml rhAng1 either alone or with 1 μM of Ang2-BD_WT, Ang2-BD_BC5, Ang2-BD_BC6, Ang2-BD_BC10, or cRGD peptide (Merck Millipore). The cells were incubated for 16–18 h at 37 °C/5% CO₂ and then washed twice in HBSS (Hanks’ balanced salt solution; Sigma). Capillary tube formation was observed using EVOS Cell Imaging Systems microscope (ThermoFisher Scientific). Images were collected with an EVOS × 2 Objective. The total number of meshes and the number of junctions of the tubes were quantified by the analysis of digitized images of the capillary-like structures using ImageJ software and the Angiogenesis Analyzer plugin.

Inhibition of adhesion of TIME cells to vitronectin was determined in 96-well microplates coated with human vitronectin (R&D Systems). Ang2-BD_WT, Ang2-BD_BC5, Ang2-BD_BC6, Ang2-BD_BC10, or cRGD peptide (Merck Millipore) (1 μM) was mixed with 5 × 10⁴ TIME cells and plated on vitronectin-coated wells either with or without 500 ng/ml of FL-Ang1, incubated at 37 °C/5% CO₂ for 2 h, and washed twice with PBS. A solution of 0.2% crystal violet in 10% ethanol was added to the wells for 10 min, which were then washed three times with PBS. Solubilization buffer (a 1:1 mixture of 0.1 M NaH₂PO₄ and ethanol) was added, and the plate was shaken gently for 15 min. Absorbance was measured at 600 nm using a microtiter plate reader (BioTek Instruments). Background signals generated with a negative control containing no cells were subtracted from the data.

The effects of Ang2-BD bi-specific variants on the growth and survival of TIME cells were assessed by an XTT assay (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay; Biological Industries). TIME cells were seeded (7500 cells per well) on a human vitronectin-coated 96-well microplate (R&D Systems) and incubated at 37 °C/5% CO₂ for 24 h. The medium was then replaced with fresh Vascular Cell Basal Medium supplemented with 2% FBS and growth factor supplements, and the cells were incubated with 500 ng/ml of rhAng1 either alone or with 2 μM Ang2-BD_WT, Ang2-BD_BC5, Ang2-BD_BC6, Ang2-BD_BC10, or cRGD peptide (Merck Millipore). The cells were incubated for 16–18 h at 37 °C/5% CO₂. Viable cells from each condition were measured by XTT at UV 450 nm, as described in the manufacturer’s protocol. The UV readings of the cell-only control were normalized to 100%, and readings from cells treated with the Ang2-BD variants were expressed as a percentage of the control.