Fibroblast proliferation and migration

FU97 cells and VASH2 knockdown (shVASH2) clones were plated in 60‐mm dishes at 5 × 10⁵ cells and cultured overnight in the culture medium. The following day, the medium was replaced by DMEM containing 0.5% FBS. The conditioned medium was collected 48 h later and filtered through a MILLEX‐GP PES 0.22‐μm filter (Millipore, Bedford, MA, USA). Cell proliferation was measured using a BrdU Cell Proliferation ELISA Kit (Abcam). Cells were plated in a 96‐well plate at 5 × 10³ cells per well and starved in DMEM containing 0.5% FBS for 16 h. Cells were then treated with conditioned media (CM) from FU97 cells or shVASH2 clones and labeled with BrdU for 24 h. Incorporated BrdU was detected according to the manufacturer's instructions. Migratory activity of fibroblasts was measured by modified Boyden chamber assay.⁶ SF‐TY cells were seeded on the upper chambers (inserts) of the Boyden chamber (8.0 μm pore size, Corning) at 2 × 10⁵ cells. The lower chamber was filled with 600 μL of CM from FU97 cells or shVASH2 clones. After incubation for 4 h, SF‐TY cells that migrated across the membrane were fixed with methanol, stained with DAPI (Sigma‐Aldrich), and counted in nine fields per insert in a blinded manner.