Assays for wound healing and cell migration

For the wound healing assay, hLECs transfected with siRNA for Arf6 were grown on 24-well plates to full confluency and starved in serum-free EBM^TM-2 Basal Medium (Lonza) overnight. Monolayers of the cells were scratched with pipette tips, and stimulated with 200 ng/ml of VEGF-C. Cell images were obtained with the Biozero BZ-X700 microscope and wound closure was analyzed by NIH ImageJ software.

For the transwell migration assay, hLECs starved as described above were seeded in the upper chamber of transwell migration chamber (8 μm pore size; Corning) at 2 × 10⁴ cells per chamber. The lower chamber was filled with 200 ng/ml of VEGF-C/EBM^TM-2 Basal Medium. At 12 hours after seeding, membrane filters were fixed with 4% PFA/PBS and stained with DAPI. hLECs migrated to the lower surface of the membrane filter were imaged using the Biozero BZ-X700 microscope, and cell number was counted.

For time-lapse analysis of wound-induced cell migration, hLECs transfected with siRNA for Arf6 were cultured on the μ-Dish dish (ibidi, Germany) to full confluency, and starved in serum-free EBM^TM-2 Basal Medium overnight. The monolayer of the hLECs was scratched with pipette tips, and then incubated in the humidified chamber of a time-lapse microscopy (FLUOVIEW FV10i, Olympus) in the presence or absence of 200 ng/ml of VEGF-C at 5% CO₂ and 37 °C. Cell migration at the wounded area was recorded every 10 minutes for 20 hours by tracking the nucleus using the manual-tracking tool of NIH ImageJ. Cell trajectories were analyzed using the Chemotaxis and Migration Tool Software (ibidi). Accumulated distance was calculated as the sum of all cell movement. Euclidean distance represents the straight distance between the start and end point of cell migration. Directionality was calculated by dividing euclidean distance by accumulated distance.