Regulation of Cell Adhesion by RA and FAKi

Cells were exposed to RA (1–10 μM), FAKi (1 μM) or their combination RA (1 μM) plus FAKi (1 μM) for 72 h. Fifteen thousand T-47D cells/well, fifty thousand LM3 cells/well, and twenty thousand HeLa cells/well were seeded into 96-well plates previously coated with 1% sterile gelatin (Sigma-Aldrich). Cells were incubated at 37 °C for 2 h. Non-adherent T-47D cells were removed by gentle washing with PBS. The attached cells were fixed with 4% paraformaldehyde and stained with 10% ethanol/crystal violet for 20 min. Images were captured and counted in ten randomly chosen fields per well using a Nikon Eclipse E200 microscope coupled to a high-resolution CCD digital camera (Nikon, Tokyo, Japan), as previously described [26 (link)]. Cell adhesion was calculated as a percentage of attached treated cells compared to untreated cells.