TWIST1 over-expression produces mesenchymal cellular changes consistent with changes in gene expression. GO analysis of gene expression predicted that TWIST1 would impact cellular phenotype and generate mesenchymal changes relevant to cell invasion. We tested this prediction in SNB19 cells using multiple cell-based assays. (A) TWIST1 inhibits cell aggregation. Representative (4×) image of cell aggregation of SNB19 Ctrl vs SNB19 TW cells (top row). Magnified (20×) sub-regions indicated by boxes in top row are shown below. (B) TWIST1 over-expression promoted adhesion to FN but not BSA-coated plates (shown as percent relative SNB19 control cells; mean ± SE). (C) Migration of SNB19 Tw cells through a filter membrane is increased 40% compared with SNB19 Ctrl cells. (D) Cell morphology, actin cytoskeleton architecture and FAK phosphorylation are altered by TWIST1. Representative (60×) photomicrographs of TWIST1 over-expressing and SNB19 control cells stained with anti-phospho-Tyr397 FAK (pFAK) antibodies (green), phalloidin-TRITC (red) and DAPI (blue). Phospho-FAK co-localization with F-actin along the border of lamellipodia in SNB19 TW and control cells is shown with large arrows and small arrows, respectively. Scale bar = 18 μm.