Depletion of mDia2 in B16F1 Cells by siRNA
(A) Staining of endogenous mDia2 by antibody (red) and actin by phalloidin (green). mDia2 is concentrated in lamellipodia and at filopodial tips (see insets at right).
(B) Western blotting of cell populations transfected with control or mDia2 siRNA. Tubulin is used as a loading control. Amount of mDia2 is decreased by approximately 60%; amounts of other proteins involved in protrusion do not decrease.
(C) Inhibition of cell spreading by mDia2 siRNA. Projected cell area was determined 2 h after cell plating. Top and bottom of a box indicate 75th and 25th quartiles, respectively; whiskers indicate 10th and 90th percentiles, respectively; dot is the mean; and the middle line is the median. An asterisk (*) indicates statistical significance (p < 0.0001, n = 120–129 cells).
(D) Inhibition of cell migration by mDia2 siRNA. An asterisk (*) indicates statistical significance (p < 0.001, n = 34–35 cells).
(E) Inhibition of lamellipodia protrusion by mDia2 siRNA. Analysis of kymographs (top) showed that rate and persistence of lamellipodia (bottom) are reduced in mDia2 siRNA-treated cells (red) compared to control siRNA (blue). Coexpression of siRNA-resistant GFP-mDia2* (purple) partially rescues the rate and fully rescues the persistence of mDia2 siRNA cells. Differences between datasets connected by brackets are statistically significant (p < 0.001, n = 20–36 cells). Red arrows in kymographs indicate the direction and the scale of time and distance.
(F) Cell transfected with mDia2 siRNA (left), in contrast to control cell (right), poorly forms lamellipodia. Numbered boxes indicate regions for the time-lapse sequences shown in (G).
(G) Frames from time-lapse sequence (25 s apart) for boxed regions in (F) (top, box 1 is shown; middle, box 2; and bottom, box 3).
(H) Immunostaining of p16-Arc subunit of Arp2/3 complex (left) and fascin (right) in control and mDia2 siRNA-treated cells. Both proteins lose their characteristic enrichment in lamellipodia and filopodia, respectively. mDia2 siRNA is shown in red.
Scale bars indicate 10 μm in (A); 10 μm in (F); and 25 μm in (H).
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