Figure 4

Influence of MMP inhibition on integrin expression levels and its membrane localization in MCF-7, MDA-MB-231 and HT-1080 cells. (A) Western blot analysis of integrin β1 in DMSO treated and GM treated cells cultured on stiff PA gels. β-actin served as a loading control (n = 2). (B) Representative maximum intensity projection images (along the height of the cells (YZ plane)) of DMSO treated and GM treated cells stained for integrin β1 (red) and DNA binding dye DAPI (blue) (Scale bar = 8 µm). Insets show localization of integrin β1 at the basal cell-ECM interface (ROI, region of interest). (C) Quantification of mean integrin β1 intensity at the basal surface in DMSO treated and GM treated cells (normalized with respect to mean intensity of DMSO treated cells) (n = 2, 25 cells per condition, ***p < 0.001, *p < 0.05). Statistical significance was determined by one-way ANOVA/Fisher test. Error bars represent standard error of mean (±SEM). (D) Quantification of membrane-localized integrins using Atomic Force Microscopy (AFM). Schematic shows probing of cells using 0.1 mg/ml RGD-functionalized spherical probes of diameter 4.5 µm. For this experiment, after indentation, tip was held in position for 10 secs (i.e., dwell time = 10 secs) to allow formation of integrin-RGD bonds, and then retracted. Representative force curve showing indentation of cell and breakage of integrin-RGD bonds during retraction. Maximum adhesive force corresponds to the maximum number of bonds formed. (E) Representative retraction curves in DMSO treated and GM treated cells cultured on stiff PA gels. (F) Histogram of maximum adhesive force in DMSO treated and GM treated cells cultured on stiff PA gels (n = 3–4, 100 cells per condition).