mDia1 is responsible for protrusions formed in N-WASP/WAVE2 double KD cells. (A) Relative expression of mDia family members in carcinoma cells. (B) Quantification of filopod formation. Cells were transfected with scrambled siRNA + YFP-vector, N-WASP/WAVE2 siRNA, and with either YFP-DN-mDia1 (NWW2 + DNmDia1) or YFP-vector (NWW2). Error bars indicate ±SEM of a total of 25 cells from three independent experiments. (C and D) Cells were treated with the following siRNAs: Scrambled (Scr); N-WASP/WAVE2 (NWW2); N-WASP/WAVE2 and mDia1 (NWW2 + mDia1); N-WASP/WAVE2 and mDia2 (NWW2 + mDia2). (C) Quantification of filopod formation. (D) Quantification of cell area fold increase during lamellipod extension. Error bars indicate ±SEM of a total of 30 cells from at least three independent experiments. (E) Western blot of cells treated with Scr, N-WASP/WAVE2, N-WASP/WAVE2+mDia1, or N-WASP/WAVE2+mDia2 siRNA and probed with anti-WAVE2, –N-WASP, -mDia1, or -mDia2 (right to left blot) and tubulin and β actin antibodies. P-values are in comparison to scrambled control.