Ecm001

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)}.

To conduct wound healing assay, cells were seeded into 12-well plates and then incubated over 90% confluence. The plate was scratched with pipette tips and washed with PBS. Cells were incubated for 24 h with fresh DMEM medium containing either vehicle alone or ODZ10117. Digital images were obtained using the Leica Application Suite (LAS) Microscope Software (Leica Microsystems).
Invasion assay was performed using a Boyden chamber system (Neuro Probe, Gaithersburg, MD, USA). Growth factor reduced Matrigel (354230, BD Matrigel™, BD Biosciences) was diluted with serum free media with ratio of 1:3. Diluted Matrigel was transferred into 24-transwell (BD 24-well insert, 8 μm pore transparent PET filter) and incubated at least for 4 to 5 h for gelling at 37 °C. Cells in 100 μL DMEM containing 1% FBS were seeded in the upper chamber and incubated for 24 h in the presence of either vehicle alone or ODZ10117. The lower chamber was filled with 500 μL of 10% DMEM containing fibronectin (5 μg/mL, ECM001, Sigma-Aldrich). Matrigel containing upper chamber was rinsed with PBS, fixed, stained with Diff-Quik solution (Sysmex Corporation, Kobe, Japan), and subsequently rinsed with distilled water. The migrated cells were captured using the LAS Microscope Software (Leica Microsystems).