Transwell migration assay

BxPC-3 and HPAC cells were cultured in serum-free medium for 24 h. Cells at 80% confluence were trypsinized. Trypsin was neutralized with medium containing 5% bovine serum albumin (BSA). The cells were resuspended in culture medium containing 0.2% BSA and were seeded (1x 10⁵ cells/well) into 8 micron transwell chambers and then VS-5584 (final concentration 0.25 μM) and SCH772984 (final concentration 0.25 μM), alone or in combination, were added to bring the final volume to 100 μL. The chambers were inserted into a 24-well plate containing 650 μL medium with 5% FBS and corresponding concentrations of VS-5584 and SCH772984, alone or in combination. The cells were incubated for 24 h, and then the cells on the surface of the top chamber were removed using cotton-tipped applicators. The cells on the bottom surface of the chamber were fixed with methanol, stained with crystal violet, and then washed twice with PBS. Images were taken using a light microscope with a 10 x objective lens. Stained cells were then eluted using 33% acetic acid and absorbance was determined at 570 nm. Results were obtained from three independent experiments. The cell migration rates, compared to control, are shown as mean ± SEM.