Figure 4
Effects of Buserelin on focal adhesion assembly and Collagen I invasion in MDA cells. A. Focal adhesion and F-actin arrangement as disclosed by confocal microscopy. MDA cells were transfected with the WT and DesK191 GnRHRs or co-transfected the WT GnRHR and GAP variants cDNAs, and exposed to 10-7M of Buserelin (upper panel) or vehicle (lower panel). The cells were then fixed and F-actin and Vinculin were stained as described in Materials and Methods. An increase in focal adhesion and stress fibers can be observed in cells overexpressing the GnRHRs as well as in those overexpressing the WT GnRHR and GAP-Y1284D (arrows in panels 2, 3 and 5), whereas cells co-transfected with the WT GnRHR and the GAP domain cDNAs exhibited decreased focal adhesion and marked accumulation of F-actin in the periphery (arrows in panels 4 and 9). Similar results were found in two other experiments. Bar: 10μm. B. The effect of GnRHR activation by Buserelin on cell invasion capacity as determined by invasion assays in Collagen-1-covered Transwell Chambers. C. F-actin levels in cell invasion. Transfected cells were attached to Collagen and exposed to Buserelin (10-7M) or vehicle in 10% FCS-supplemented medium, and the amount of F-actin was determined by fluorometry. The results shown in B and C are the means ± SEM from 3 independent experiments. *p<0.05; **p< 0.01; ***p< 0.001.