Crystal violet adhesion assay

SW480 and SW620 cells were seeded at a density of 1.2 x 10⁵ cells/well into a 96-well plate and treated with combinations of TGF-β1, Hsp90β, SB431542, novobiocin and αvβ6 integrin blocking antibody as indicated in figure legends. The cells were left to adhere for 8 h at 37 °C before removing the media and washing the wells with PBS three times. Adherent cells were fixed with 4% [v/v] paraformaldehyde in PBS before washing with deionised water and staining with 10% [w/v] crystal violet in 5% [v/v] ethanol. Wells were again washed with deionised water, allowed to air-dry and crystal violet dye solubilised in 5% [w/v] SDS and 1% [v/v] Triton-X100. Absorbance was read at 590 nm using a microtitre plate reader (PowerWaveXTM, BioTek).