Supplementary Figure 4: Temporal regulation of MLC and Ezrin.

(a,b) Serum stimulation of serum starved A431-MLC-GFP (a) and A431-Ezrin-GFP cells (b) induces an immediate translocation of MLC-GFP and Ezrin-GFP to the actomyosin network at the outer edge (white arrows) of A431 cell clusters and at cell-cell junctions (red arrows). Quantification of the serum-induced MLC-GFP and Ezrin-GFP translocation +/−s.e.m was performed by normalizing the mean fluorescence intensity at cell border (left graph) and cell-cell junctions (right graph) to the mean fluorescence intensity of the adjacent cytosolic fraction. n = regions of individual measurements (63x) at edge of cluster and cell-cell contacts; MLC, 70; Ez, 35. The values are pooled from three independent experiments. (c) F-actin staining of serum starved A431 cells and after 5 minutes of FCS stimulation. FCS stimulation induces slight blebbing at the cortex of the cells. Blebs are indicated by white arrows. All experiments were conducted at least 3 independent times. Scale bars, 10 μm.