RhoA activity is increased in N-WASP/WAVE2 KD cells and is required for filopod production in N-WASP/WAVE2 double KD cells. (A–F) MTLn3 cells were transfected with scrambled control, WAVE2, N-WASP, N-WASP/WAVE2, or Arp2/3 siRNA as indicated in blots and graphs. (A and B) The cells were then starved for 3 h and stimulated with EGF for the indicated times. (A) Representative Western blots of GST-RBD pulldowns showing Rho activation in EGF-stimulated MTLn3 cells. Proteins bound to immobilized GST-RBD were immunoblotted with anti-RhoA,B,C antibody. The amount of total Rho in cell lysates is shown at the bottom. (B) Quantification of the Western blot data by densitometry (arbitrary units) for RhoA in A. P-values are in comparison to control at each time point or in comparison to the zero time point (brackets). Activation is calculated as percent of the maximum within each experiment. Error bars indicate ±SEM of three independent experiments (C) Western blot representative of RhoA pulldown (top) and total cell lysate (bottom) of cells treated with scrambled and Arp2/3 siRNA at steady state. (D) Quantification of the Western blot data by densitometry (arbitrary units) for RhoA in C. (E) Inhibition of RhoA results in a decrease in the filopods produced in N-WASP/WAVE2 KD cells. Quantification of filopod formation. Cells were transfected with either N-WASP/WAVE2 or scrambled siRNA for 48 h. N-WASP/WAVE2 siRNA cells were treated with (NWW2+C3) or without (NWW2) tat-C3 toxin. Error bars indicate ±SEM from a total of 24 cells from three independent experiments. (F) Dominant-active RhoA increases filopod formation in MTLn3 cells. Quantification of filopod formation. Cells were transfected with either N-WASP/WAVE2 or scrambled siRNA for 48 h. N-WASP/WAVE2 siRNA cells were also transfected with CFP or CFP-DA-RhoA (NWW2+DA-RhoA). Error bars indicate ±SEM, from a total of 21 cells, from three independent experiments.