Fig. 2. Cdc42-siRNA-treated cells show reduced protrusion in response to EGF stimulation. (A) Representative western blot showing Cdc42 (upper panel) and Rac (lower panel) expression in cells treated with control luciferase-siRNA-treated (Luc) or Cdc42 siRNA (Cdc42 KnDn). Quantification of western blots from four different experiments (bar graph) shows an 80% decrease in the level of expression of Cdc42 in cell lysates. (B) Representative phase micrographs of MTLn3 cells transfected with control luciferase or Cdc42 siRNA (Cdc42 KnDn), starved and stimulated with EGF for 1 or 3 minutes or not (0 min). (C) Quantification of the surface area of EGF-stimulated control luciferase-siRNA-treated () and Cdc42-siRNA-treated cells () normalized to unstimulated cells. Data are the mean ± s.e.m. from at least 45 cells. Bar, 10 µm. (D) MTLn3 cells were transfected with luciferase control or Cdc42 siRNA and starved for 3 hours. Cells were then imaged using time-lapse microscopy during a 10-minute EGF stimulation with images collected every 20 seconds. Kymographs were generated by drawing a constant line in the stacked time series and the protrusion was followed over time.