VSMC Migration Assay

The wound healing assay was performed to test migratory capability of VSMCs. Briefly, VSMCs were seeded into 24-well tissue culture plates. The cells were incubated in DMEM supplemented with 0.5% FBS for 24 h to reach 70–80% confluence. The cell layer was scratched gently with a new 200 μL pipette tip across the center of the well. The cells were washed three times in 1 × PBS to remove the detached cells and incubated in DMEM supplemented with 0.5% FBS. The cells were treated with TNF-α (100 U/mL) in the absence and presence of PPP or PU for additional 24 h, and then washed twice in 1 × PBS and finally stained with 1% crystal violet in 2% ethanol for 30 min. Photos were taken for the stained monolayer on a microscope. Multiple views of each well were documented, and each experimental group was repeated 5 times.