N-WASP mediates leading cell collective invasion into 3D matrices. (A) Cells that migrated into Matrigel plugs in an inverted invasion assay were stained with CalceinAM and visualized by confocal microscopy. Serial optical sections were captured at 15-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. The assays were quantified by measuring the fluorescence intensity of cells penetrating 30 µm and greater. 0 µm indicates cells that crawled through the filter but did not enter the gel. The invasion capacity was expressed as a percentage of the total fluorescence intensity of all cells within the plug, as shown in the bar graph. At least three independent experiments were performed. All error bars indicate means ± SD; **, P < 0.01 by a t test. Bar, 100 µm. (B) Matrigel plugs containing cells from inverted invasion assays were fixed and stained with phalloidin (actin, red) and DAPI (DNA, blue). Strands of invading cells are shown in cross section and side views. Bar, 50 µm.