Fig. 4
From: The NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based WAVE2 complex
NESH/Abi-3 promoted invadopodia formation in the presence of imatinib. (a) Lysates were prepared from SRD cells, and SRD cells expressing Abi-1, FLAG-Abi-2, or NESH. Western blotting analysis was performed with the indicated antibodies. (b) The wound-healing assay was performed, and the migration area was quantified as described under Materials and Methods. Data represent the means ± S.D. for three independent experiments. Error bars represent S.D. The value for the control SRD cells was set to 100 %. *, P < 0.05; ***, P < 0.001, Student's t test. (c) The indicated cells were plated onto TRITC gelatin-coated dishes. After 7 h, cells were fixed and stained with an anti-cortactin antibody. Cells with a punctate cortactin-rich structure were counted under a fluorescence microscope. At least 100 cells were analyzed for each sample. Data represent the means ± S.D. for three independent experiments. Error bars represent S.D. **, P < 0.005; ***, P < 0.001, Student's t test. (d) Invadopodia degradation areas were quantified as described under Materials and Methods. For each sample, five cells exhibiting invadopodia degradation were randomly picked up and the degradation area/cell area was calculated. Data represent the means ± S.D. for three independent experiments. Error bars represent S.D. **, P < 0.005; ***, P < 0.001, Student's t test. (e) Representative confocal microscopy images of SRD cells, and SRD cells expressing Abi-1, FLAG-Abi-2, or NESH in (D). The black dots indicate the invadopodia-mediated gelatin degradation. (f, g) SRD cells and SRD cells expressing Abi-1, FLAG-Abi-2, or NESH were treated with the c-Abl siRNA for 72 h, and then analyzed as described in (c) and (d). (f) The percentage of cells containing a punctate cortactin-rich structure. (g) Quantification of the degradation area. Data represent the means ± S.D. for three independent experiments. Error bars represent S.D. *, P < 0.05; **, P < 0.005; ***, P < 0.001; n.s., the difference was not significant (P = 0.062 in (f), P = 0.17 in (g)), Student's t test