Fig. 2.

Eps8 regulates polarized cell migration and invasion. FAK WT and FAK^−/− cells were transiently transfected with two independent Eps8 siRNAs (siEps8 #1 and #2; siC, control siRNA) and (A) lysed 48 h post transfection and Eps8 expression determined by western blotting using anti-Eps8 antibody. Anti-β-actin was used as a loading control. (B) Confluent monolayers of cells plated on fibronectin were wounded using a pipette tip. Wound closure was quantified at 15 h post wounding. Results are mean±s.e.m. *P<0.01 (Student's t-test). (C) Confluent monolayers of cells plated on fibronectin were wounded using a pipette tip, fixed 3 h later and stained with anti-GM130 antibody (to label the Golgi) and TRITC–phalloidin. Polarization was scored using the orientation of the Golgi towards the wound edge. Solid arrows indicate polarized cells. Dashed arrows indicate unpolarized cells. Scale bars: 20 µm. Results are mean±s.d. *P<0.001 (Student's t-test). (D) Cells were seeded on growth-factor-reduced Matrigel in serum-free conditions. Invasion towards a serum gradient (the distance of the horizontal z-section from the top of Matrigel is given) was visualized after 72 h by staining the cells with calcein. Results are mean±s.e.m. *P<0.001 (Student's t-test). Quantification is representative of at least three independent experiments in all cases.