24 well transwell with 8 μm pores

The transwell assay was performed as described previously [28 (link)]. KYSE150 and KYSE510 cells were starved in serum-free medium for 12 h after being transfected. A total of 5 × 10⁴ cells were plated in medium without serum in the upper well of a transwell chamber of a 24-well transwell with 8 μm pores (BD Biosciences), which was placed in a bottom chamber containing medium supplemented with 10% fetal bovine serum. After 48 h, the membranes were fixed and stained with haematoxylin solution and scraped off the cells remaining in the upper chamber. The migration was quantified by counting 10 random fields under a Leica DMI3000B inverted phase-contrast microscope (400) with Image J. Each experiment was performed in triplicate.

Transwell assay was performed as described previously (Zhang et al., 2018c). KYSE150, KYSE450, and TE3 cells were starved in serum‐free medium for 12 h after being transfected. A total of 5 × 10⁴ cells were plated in medium without serum in the upper well of a transwell chamber of a 24‐well transwell with 8‐μm pores (BD Biosciences, San Jose, CA, USA), placed in a bottom chamber containing medium supplemented with 10% FBS. After 48 h, the membranes were fixed ice‐cold methanol and stained with hematoxylin solution, and migration was quantified by counting 10 random fields under a Leica DMI3000B inverted phase‐contrast microscope (400×). The migration cell numbers were counted with imagej. Each experiment was performed in triplicate.