Cell migration and invasion assays

For transwell migration experiments, cells were washed with serum-free media, and 20,000 cells were plated in replicate in 200 μl of serum-free media in the top of the well insert (8 μm pore, BD Biosciences, #353097). Inserts were then placed in a 24-well companion plate with 600 μl of media containing 10% fetal bovine serum (FBS) as a chemoattractant. After incubation for 16 hours, cells were fixed for 20 minutes in 70% ethanol, and unmigrated cells were removed by cleaning the top of the membrane with a cotton swab. Migrated cells were permeabilized for 5 minutes in 0.3% Triton X-100 in PBS. Cells were stained with 3 μg/ml DAPI in PBS for 20 minutes. Five random fields at 10× power were taken of each well. Quantitation was carried out using the Nikon NLS software object count. Transwell invasion experiments were carried out in a similar manner using Matrigel Invasion Chambers (Corning, #354480) and incubation for 36 hours.