Figure 1. TβRIII mediates epithelial cell adhesion to fibronectin, regulates focal adhesion formation and FAK activation in response to spreading on FN.

MCF10A mammary epithelial cells adenovirally infected with either shTβRIII-1 or shTβRIII-2 to human TβRIII in parallel with controls (shNTC or shCtl) were (A) assessed for cell surface expression of TβRIII by ¹²⁵I-TGF-β binding and crosslinking, and for total cellular β-arrestin2, fibronectin and integrin α5 expression, with β-actin as a loading control, and (B) assessed for adhesion to the indicated concentrations of FN for 30 minutes and the adhesion level plotted (mean+/−SD ,*p<0.001). Adhesion assays were performed as described in Methods with BSA as a negative control. (C) MCF10A cells were infected with adenovirus expressing shTβRIII-1, control (shNTC), or TβRIII (HA-TβRIII), plated on FN coated cover slips for the indicated times, FAs were detected by anti-vinculin antibody against endogenous vinculin using TIRFM. (D) FA length and the mean +/− SEM were plotted relative to the respective controls (shNTC for shTβRIII; endogenous TβRIII for HA-TβRIII), * p<0.01. Length and intensity of 10 visibly distinct focal adhesions/cell was measured working clockwise from the top of the cell for 10 cells (100 FA measurements per condition). (E) MCF10A-shTβRIII-1 (left panel) or MCF10A-shTβRIII-2 (right panel) with MCF10A-NTC cells as controls were spread on 5 μg/ml FN coated wells for the indicated times and FAK activation (pFAK at Tyr 397), determined by western blot with total FAK as a loading control. Quantitation of the amount of pFAK relative to total FAK is presented below. (F) MCF10A cells transiently overexpressing either TβRIII or β-arrestin2 were spread on 5 μg/ml FN coated wells for the indicated times and FAK activation (pFAK at Tyr 397), determined by western blot with total FAK as a loading control. Quantitation of the amount of pFAK relative to total FAK is presented below.