Cell Migration and Invasion Assay

We detected cell migration and invasion using Transwell assay as described previously (14 (link)). In total, 5×10⁴ cells/well in serum-free medium were seeded into the top chamber (pore size, 8 µm; Corning Incorporated, Corning, NY, USA). For the cell invasion assay, the upper chamber was coated with Matrigel (1:8; 50 µl/well; BD Biosciences, San Jose, CA, USA) and the cell number is 8×10⁴ cells/well for 769-P. The bottom chamber was filled with conditioned medium with 10% FBS. After 24 h incubation, the cells were fixed with 4% paraformaldehyde and stained using 0.1% crystal violet for 5 min at room temperature. Images were captured using an inverted microscope.