Human vitronectin

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.

G-rhChM-I was expressed in Chinese hamster ovary (CHO) cells and purified from culture supernatants as described previously [6] (link). NG-rhChM-I was expressed in E. coli BL21 and purified as described [13] (link). The disulfide pairings were confirmed by trypsin digestion and peptide mapping of disulfide-bridged G-rhChM-I and NG-rhChM-I [6] (link), [13] (link). The N-terminal deleted 14-kDa rhChM-I (ΔN-rhChM-I) was prepared by incubation of NG-rhChM-I with V8 protease (Pierce, Rockford, IL), and the resultant products were then purified by Butyl-Toyopearl 650 M (Tosoh, Tokyo, Japan) and reversed-phase HPLC [13] (link). Recombinant human vascular endothelial growth factor-A₁₆₅ (VEGF-A₁₆₅) and human vitronectin were purchased from R&D Systems (Minneapolis, MN) and BD Biosciences (Bedford, MA), respectively. Other chemicals were purchased from Sigma (St Louis, MO).

Cell adhesion assays were performed as previously described
[30 (link)]. The wells of 96-well Maxisorp plates (Nunc) were coated overnight at 37°C with either 1% BSA (Bovine serum Albumin, Sigma), 2.5 μg/ml human Vitronectin (R&D) or recombinant human Netrin-4 (R&D) diluted in PBS. After two washes in PBS, non-specific binding sites were blocked for 1 hour at 37°C using 1% BSA. After washes with PBS and water, 100 μl of a cell suspension containing 500000 cells per ml in culture medium was added (three wells per treatment) to each well and incubated at 37°C for 4 hours. Non-adherent cells were washed off with water. Cells that adhered to the substrate were fixed and stained with crystal violet (0.2% in methanol). Images were acquired with an inverted microscope (Nikon Eclipse Ti) equipped with a digital camera. Dye bound to adhered cells was solubilized with 0.1% SDS and the absorbance at 560 nm was measured. The data reported were mean values of the three determinations per treatment.