Figure 3

Fascin overexpressed cells demonstrated increase in cell-ECM adhesion and loss of cell-cell contacts. (A) Cell adhesion of fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells to different ECM substrates was measured as described. The data shown is the average from three experiments with the mean and standard deviation. (B) Western blot analysis of stable fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) clones with antibodies to α6 integrin, β4 integrin and phosphorylated FAK. β-actin and total FAK were used as internal loading controls respectively. (C) Representative images of aggregates formed by of fascin overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells in hanging drop assay. (D) Histogram showing size of aggregates formed by overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells. Mean and standard deviation of 3 independent experiments is plotted (p < 0.0001). (E) Confocal analysis of E-cadherin and β-catenin localization in fascin overexpressed cells. Scale bars; 10 μm. (F) Western blot analysis of fascin overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control clones (AW-GFP-Cont) with antibodies to E-cadherin and β-catenin. β-actin was used as a loading control. (G) RT-PCR analysis of snail, slug and vimentin overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control (AW-GFP-Cont) cells. GAPDH was used as internal control.