Cells were fixed with 4% paraformaldehyde for 10 min and permeabilised in 0.1% Triton X-100 for 10 min. Cells were blocked in 1% BSA for 1 hr before incubation with primary antibodies – pS19-MLC (Cell Signaling #3671 L), myosin MHC IIa (Covance PRB-440P), fibronectin (Sigma F3648), β-catenin (Santa Cruz sc7963), integrin β1 (Santa Cruz sc13590), and integrin β3 (Abcam, ab179473) at 4°C overnight. After incubation, the appropriate fluorescence-conjugated secondary antibodies for 1 hr, cells were washed with PBS. Images were acquired with an inverted Zeiss LSM780 at a magnification of ×20 and ×63. For quantification of the pMLC staining, regions of interest were drawn around equal numbers of ‘free boundary zones’ of A431 cells in clusters and cell-cell contact zones, and the mean fluorescent intensity was measured. The values were then normalised to the mean of all the boundary and contact zones for WT A431 cells. Staining of frozen human tissue sections was performed in a similar manner, except that fixation and permeabilisation times were doubled, and 5% BSA was used as a block.
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