Migration and invasion assays

These assays were performed on cells 48 hours after transfection and 24 hours after starvation. Briefly, cells were resuspended with serum-free medium, then 3×10⁴ A498 and 20×10⁴ Caki-1 cells added to the upper chamber of the transwell for analysis of migration. To assess invasion, 6×10⁴ A498 and 40×10⁴ Caki-1 cells were seeded in chambers where Matrigel (Thermo Fisher Scientific; Waltham, USA) had been added in advance. After 24 hours (A498 cells) or 48 hours (Caki-1 cells) incubation, the invaded cells were fixed in 100% methanol, then stained with 0.05% crystal violet before counting of 5 randomly selected fields.