Cell migration assay

Cell migration ability was measured by scratch assay and Transwell assay. For the scratch assay, cells were seeded into six-well plates and incubated until they reached ~90% confluence. Clear lines in the wells were created using a sterile 200 µL pipette tip. A digital camera system was used to quickly take photographs of each well immediately after scratch creation, and then 24 hours later, the same areas were re-photographed. Migration distance was measured at the time points of 0 and 24 hours using the software program HMIAS-2000. For the Transwell assay, Transwell inserts with 8.0 µm pores (COStor, Kennebunk, ME, USA) were placed into wells of a 24-well plate. Cells were first starved in 200 µL serum-free medium and then 3×10⁴ cells were placed into wells of the uncoated dishes. The bottom chambers were filled with 500 µL medium supplemented with 10% FBS. Cells were cultured at 37°C in a humidified atmosphere with 5% CO₂ for 24 hours, after which the cells that had migrated to the bottom surface of the filter membrane were fixed in 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet (Sigma-Aldrich Co.) for 25 minutes. The superfluous dye on the surface of the polycarbonate membrane was removed with sterile double-distilled water (ddH₂O) and a cotton swab. The invaded cells were observed and photographed using an inverted microscope. Next, the chambers were soaked in 1 mL of 33% glacial acetic acid for 10 minutes to wash out the crystal violet. Then, 100 µL of 33% acetic acid was added to each well of the 96-well plate and the absorbance was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad Laboratories Inc.). All experiments were performed at least three times.