Cell Migration and Colony Formation Assay

After RP11-366H4.1.1, LINC460 or AC093850.2 was subjected to individual knockdown, KYSE150 or KYSE70 cell migration and colony formation assays were performed as previously described [16 (link)]. Briefly, at 24 h post transfection, cells were starved for 12 h with serum-free medium (Invitrogen, California, USA) and then 5 × 10⁴ cells were plated in serum-free medium in the upper well of a transwell chamber (24-well insert; pore size, 8 μm; BD Biosciences, Franklin Lakes, NJ, USA), and the lower chamber containing medium with 10% FBS. After 48 h, cells in the top chamber were removed with a cotton swab and only cells that migrated through the pores were fixed and stained in haematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA) and counted. For colony formation, 500 cells per well in 24-well plate were incubated in medium supplemented with 10% FBS for ten days, and then colonies were stained with haematoxylin solution and observed.