hGAAP expression alters the number and size of focal adhesions. U2-OS cells transfected with siRNA (24 h) or plasmids encoding hGAAP were seeded onto fibronectin-coated slides. (A) Confocal images show cells transfected with vinculin-GFP (14 h), fixed, and stained with phalloidin–Alexa Fluor 568. (B) Summary results (means ± SEM from ≥25 cells for each condition) show numbers of focal adhesions per cell, determined by counting vinculin-GFP spots. (C) IRM images of U2-OS cells overexpressing hGAAP or hGAAP Ctmut, or transfected with the indicated siRNAs. Images are typical of three independent experiments. (D) Example of the image analysis used to determine focal adhesion number and size. Individual cells were imaged for IRM (a) and phalloidin staining (b). IRM images were then band pass filtered (c), and a threshold was imposed (d) to obtain the final images used to determine the number and size of the adhesions. (E and F) Summary results (means ± SEM from ≥20 cells for each condition) show numbers of adhesions per cell (E) and their areas (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test). Bars, 10 µm.