mDia1 localization and activity. (A) Localization of mDia1 by antibody staining in N-WASP/WAVE2 double KD cells and control cells. Cells were treated with Scrambled or N-WASP/WAVE2 siRNA and stained with anti-mDia1 antibody (green) at 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (red). Insets show actin protrusion with filopods in N-WASP/WAVE2 KD cells. Bar, 10 μm. (B) Constitutively active mDia1 activation gives similar phenotype to N-WASP/WAVE2 double KD cells. Images of cells transfected with control GFP empty vector (top, red) or GFP-ΔGBD-mDia1 (bottom, red), a DA mDia1, for 6 h. Images were taken of cells fixed at 0, 1, and 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (green). Insets (indicated by dashed boxes) show active mDia1 localized at tips of filopods. Bar, 10 μm. (C) Quantification of filopod formation. P-values are compared with control GFP vector cells. (D) Total F-actin fluorescence intensity (percent) was measured using the mean value of phalloidin staining of unstimulated cells transfected with GFP or DA-mDia1 vector. P-values are in comparison to control. Error bars indicate ±SEM of a total of 19 cells from three independent experiments.