Transwell migration and invasion assays were implemented using Transwell chambers (Corning, Inc.). Chambers were pre-coated with or without Matrigel (Corning, Inc.) at 4°C overnight. HUVECs were collected at 24 h post-transfection, resuspended in fresh culture medium with serum depletion, and transferred into the top of chambers (5×104 cells) coated with or without Matrigel for invasion or migration analysis, respectively. The bottom chambers were filled with fresh culture medium containing 10% FBS to induce cell migration and invasion. Allowing for migration and invasion for 24 h at 37°C, the cells that had migrated or invaded into the lower surface were fixed with formaldehyde, stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 20 min at 37°C and then observed under a CKX41 light microscope (Olympus Corporation) at a magnification of ×100.

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