Tube Formation Assay for Angiogenesis

The tube formation assay was performed as previously described^{8 (link)}. Briefly, endothelial cells were seeded at a density of 1–2 × 10⁴ cells/well for 6 h in plates precoated with Matrigel at 37 °C and 5% CO₂. For coculturing experiment, LX-2 cells were seeded into the upper chambers of a transwell plate (Corning, Costar 3422) for 24 h and then transferred to other plates with endothelial cells. Experiments were performed in the presence or absence of various fibronectins, including pFN (40 μg/ml, ECM001, Sigma-Aldrich; St. Louis, MO, USA), FN-EDA (40 μg/ml, F2518, Sigma-Aldrich; St. Louis, MO, USA), rEDA (40 μg/ml Flag-tagged, Daian, Wuhan, China) or inhibitors including irigenin (5 μM Selleck, Shanghai, China) and, resatorvid (5 μM, MCE, USA), or neutralizing antibodies (Table 4). Tube formation was photographed using inverted microscope and quantified by calculating the average tube length using ImageJ software (National Institutes of Health, Bethesda, MD, USA)^{58 (link)}.