Scratch and Transwell® migration assays

For the scratch migration assay, cells (2×10⁴ cells/well/100 µl) were seeded into 96-well plates, allowed to adhere overnight and treated with 25 µg/ml mitomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min. Following the creation of wounds on the confluent monolayers using a 96-pin Wound Maker (IncuCyte; Essen Bioscience, Ann Arbor, MI, USA), the plates were placed into the IncuCyte chamber (Essen Bioscience) and incubated with or without RA in a 5% CO₂ incubator at 37°C. The wound images were captured every 3 h using an IncuCyte Zoom system (Essen Bioscience). The wound closure proportion at each time point was calculated based on the wound width at 0 h as 100%. For the Transwell migration assay, a Transwell chamber (10 mm diameter, 8 µm pore size polycarbonate membrane; Corning Incorporated) was used. In the lower chamber, 600 µl 10% FBS in DMEM or EGM-2 was used for tumor cells or HUVECs, respectively. In the upper chamber, cells (1×10⁵ cells/well) suspended in 100 µl serum-free DMEM or endothelial cell basal medium-2 (EBM-2) were added. Following incubation at 37°C with 5% CO₂ for 12 h, the migrated cells were fixed, stained with 0.2% crystal violet/20% (w/v) methanol solution, and counted using a phase-contrast microscope (magnification, ×100).