N-WASP is required for path generation during invasion. (A) Equal numbers of RFP-expressing cells transfected with either NT or N-WASP siRNA (red) were mixed with GFP-labeled cells treated with NT siRNA (green) in CIA. Cells were fixed and stained for DNA (blue) to show path-generating and following cells in the invading strands. Arrowheads indicate leading cells of invasive cell strands. Arrows indicate the direction to the wound edge. At least three independent experiments were performed and quantified. Bar, 10 µm. (B) Bar graphs indicate the percentage of leading and total cells of GFP- and RFP-positive cells. Error bars indicate means ± SD; **, P < 0.01 by t test. (C) A similar setup was used as in A, except that NT or N-WASP siRNA RFP-expressing cells alone were seeded for inverted invasion assay. Serial optical sections were captured at 10-µm intervals and presented as a sequence in which the individual optical sections are placed alongside one another with increasing depth from left to right as indicated. Images at 0 µm indicate cells that came though the filter but did not enter the gel. Invasion capacity was expressed as a percentage of the total fluorescence intensity of all cells invading beyond 30 µm within the plug as shown in the bar graph. At least three independent experiments were performed and quantified. Bar, 100 µm.