N-WASP is required for extension of elongated matrix degrading pseudopods and propulsion through dense matrix. (A) MDA-MB-231 cells treated with siRNA NT and N-WASP invading under Matrigel (photos show 5 mg/ml Matrigel, bar graphs show concentrations as indicated) in a CIA. Black lines indicate the start of the cell front at t = 0 h. Cell migration speed and persistence in CIA are shown in the bar graph. Persistence is obtained by using the Euclidean distance divided by the total distance between the start and end points of cell movement. At least three independent experiments were performed and quantified. Error bars indicate means ± SD; **, P < 0.01 by t test. Bar, 100 µm. (B) Cells invading in CIA were fixed and stained with phalloidin (actin, red) and DAPI (DNA, blue) for confocal imaging. The length of actin pseudopods and the ratio of cell length/width in CIA are shown in the bar graph. Error bars indicate means ± SD; **, P < 0.01; *, P < 0.05 by t test. At least 30 cells were analyzed in three independent experiments. Bars, 20 µm.