Scratch wound healing assay

HUVECs were cultured to approximately 90% confluence and then scratched with a sterile pipette tip to create a wounded area lacking ECs. Scratched HUVEC monolayers were incubated in medium containing vehicle or TKI in 4% bovine growth serum (BGS, Cytiva Life Sciences) with 2 μM 5-ethynyl-2’-deoxyuridine (EdU, Thermo Fisher). After 18 hours cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.1% Triton in PBS for 30 minutes at room temperature, and stained with Click-iT Alexa 488 and Hoechst DNA counterstain according to manufacturer’s instructions (Click-iT EdU Alexa 488 Imaging Kit, Thermo Fisher). Cells were imaged at 100x with a Nikon Ti Eclipse Microscope with Coolsnap EZ fluorescent camera and the total number of cells, the number of EdU+ cells (proliferated cells) and the number of EdU- cells (migrated cells) in the wound were counted by a blinded investigator in 4 fields per well and averaged for each well. 5–10 independent experiments were performed per condition.