Ps19 mlc

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.

MDA-MB-231 cells were seeded in an IBIDI-μSlide (IBIDI, Martinsried, Germany) and cultivated overnight plus treatment period. F-actin was stained with rhodamin-phalloidin (1∶400, R 415, Molecular Pobes/Invitrogen) and nuclei with bisBenzimide H33342 (Sigma-Aldrich, St. Louis, MO, USA). The following antibodies were used: Integrin α5 (Millipore, Upstate), p(S19)-MLC (Cell Signalling Technology, Danvers, MA, USA) and Vinculin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Images were obtained with a Zeiss LSM 510 META (Zeiss, Oberkochen, Germany) or Leica TCS SP8 SMD confocal microscope (Leica, Mannheim, Germany).