Figure 5.
Figure 5. The simultaneous suppression of both WAVE2 and N-WASP results in a new type of protrusion during EGF stimulation. (A) Representative images of GFP–β actin–expressing MTLn3 cells treated with scrambled siRNA or double transfected with WAVE2 and N-WASP siRNA at 0 and 3 min after EGF addition. Bar, 10 μm. Side panels show a time-lapse sequence of filopod formation in an N-WASP/WAVE2 KD cell. An electron microscopy image, which is representative of the structure of actin in a filopod like that shown in the time lapse, is shown at the bottom right. The large image at the bottom is an enlargement of this. Bar, 1 μm. (B) Quantification of cell area fold increase during lamellipod extension. Cell area was measured at 0 and 3 min after EGF stimulation. Bar graph shows scrambled and N-WASP/WAVE2 siRNA. (C) Quantification of filopod formation after EGF. Error bars in B and C indicate ±SEM of a total of 30 cells from three independent experiments. (D) Filopods in the absence of EGF. P-values are in comparison to control. Error bars are ±SEM of a total of 36 cells from three independent experiments. (E) Stress fiber density (measured as total F-actin fluorescence intensity) was analyzed by measuring phalloidin staining in fixed cells. Error bars are ±SEM of a total of 58 cells from three independent experiments. (F) Western blot representative of scramble (Scr) and N-WASP/WAVE2 (NWW2) KD blotted with N-WASP, WAVE2, and actin antibodies. (G–J) Cells were treated with either p34-directed siRNA (Arp2/3si), scrambled siRNA, or p34 plus mDia1 siRNA. (G) Images representative of cells stimulated with EGF at 0 and 3 min. Bar, 10 μm. (H) Western blot of cell lysates treated with P34 (Arp2/3 si) or scrambled (Scr) siRNA and blotted with α-Arp3 and α–β actin antibodies. (I) Quantification of cell area fold increase during lamellipod extension. Cell area was measured at 0 and 3 min after EGF stimulation. Graph shows scrambled and Arp2/3-directed p34 siRNA. (J) Cells were treated with scrambled, p34, or p34 + mDia1 siRNA. Quantification of filopod formation. Error bars are ±SEM of a total of 32 cells from three independent experiments.

The simultaneous suppression of both WAVE2 and N-WASP results in a new type of protrusion during EGF stimulation. (A) Representative images of GFP–β actin–expressing MTLn3 cells treated with scrambled siRNA or double transfected with WAVE2 and N-WASP siRNA at 0 and 3 min after EGF addition. Bar, 10 μm. Side panels show a time-lapse sequence of filopod formation in an N-WASP/WAVE2 KD cell. An electron microscopy image, which is representative of the structure of actin in a filopod like that shown in the time lapse, is shown at the bottom right. The large image at the bottom is an enlargement of this. Bar, 1 μm. (B) Quantification of cell area fold increase during lamellipod extension. Cell area was measured at 0 and 3 min after EGF stimulation. Bar graph shows scrambled and N-WASP/WAVE2 siRNA. (C) Quantification of filopod formation after EGF. Error bars in B and C indicate ±SEM of a total of 30 cells from three independent experiments. (D) Filopods in the absence of EGF. P-values are in comparison to control. Error bars are ±SEM of a total of 36 cells from three independent experiments. (E) Stress fiber density (measured as total F-actin fluorescence intensity) was analyzed by measuring phalloidin staining in fixed cells. Error bars are ±SEM of a total of 58 cells from three independent experiments. (F) Western blot representative of scramble (Scr) and N-WASP/WAVE2 (NWW2) KD blotted with N-WASP, WAVE2, and actin antibodies. (G–J) Cells were treated with either p34-directed siRNA (Arp2/3si), scrambled siRNA, or p34 plus mDia1 siRNA. (G) Images representative of cells stimulated with EGF at 0 and 3 min. Bar, 10 μm. (H) Western blot of cell lysates treated with P34 (Arp2/3 si) or scrambled (Scr) siRNA and blotted with α-Arp3 and α–β actin antibodies. (I) Quantification of cell area fold increase during lamellipod extension. Cell area was measured at 0 and 3 min after EGF stimulation. Graph shows scrambled and Arp2/3-directed p34 siRNA. (J) Cells were treated with scrambled, p34, or p34 + mDia1 siRNA. Quantification of filopod formation. Error bars are ±SEM of a total of 32 cells from three independent experiments.

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