Fig. 4. Cdc42 meditates the translocation of Arp2/3 to the protruding edge in response to EGF stimulation. MTLn3 cells were transfected with control or Cdc42 siRNA, starved for 3 hours and stimulated with EGF for 1 or 3 minutes or not. The cells were then fixed and immunostained with anti-Arp3 and anti-tropomyosin antibodies. (A) Representative merged images of control luciferase-siRNA-treated (left panels) or Cdc42-siRNA-treated (right panels) cells stimulated for 3 minutes and stained for Arp3 (red) and Tropomyosin (green). Small panels show magnification of boxed areas of respective large panel. Arrows indicate the edges of Arp2/3 (red) and tropomyosin-rich (green) zones. (B) Quantification of average fluorescence pixel intensity at the edge for Arp2/3 in control luciferase-siRNA-treated (top diagram) and Cdc42-siRNA-treated (bottom diagram) cells stimulated with EGF for 1 minute ( and , respectively) or 3 minutes ( and , respectively) or left untreated ( and , respectively). Data are the mean ± s.e.m. from three different experiments (15 cells per experiment) plotted as a function of distance from the cell edge. (C) Quantification of average fluorescence pixel intensity at the edge for tropomyosin in control (top diagram) and Cdc42 siRNA-treated cells (top diagram). Cells were stimulated as in B. (D) Data from B presented as an average of the mean fluorescence in the lamellipodia (0.2-0.6 µm from the edge) normalized to unstimulated cells. Bar, 10 µm.