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Fig. 3. BMP2-induced actin reorganization and cell migration is mediated by Cdc42. (A) Swiss 3T3 cells were serum-starved for 7 days and stimulated with 3 nM BMP2. Arrows indicate spike-like filopodia (see detail at 30 minutes). As a control, cells were stimulated with EGF 20 ng/ml for 10 minutes (upper row). Scale bar, 20 µm. (B) Serum-starved C2C12 cells were stimulated with 3 nM BMP2. Levels of active GTP-bound Cdc42 and Rac were determined using the PBD domain of PAK1 followed by immunoblotting with anti-Cdc42 and anti-Rac antibodies. (C) C2C12 cells were transfected with the indicated GFP-tagged expression vectors. Cells were serum-starved for 16 hours (control), pre-treated with cytochalasin D and allowed to recover for 1 hour in the absence or presence of 3 nM BMP2. Merged images of phalloidin (red) and GFP signal (green) are shown. Cells showing cortical actin protrusions (arrows) are indicated. Scale bar: 20 µm. Transfected cells were counted and those showing cortical actin protrusions represented as % of total (graph on right). Mean ± s.e.m. of at least 80 transfected cells obtained from three independent experiments (*P<0.001, paired t-test). (D) C2C12 cells were transfected with the GFP-tagged expression vectors indicated and analyzed by chemotaxis assay after 2 hours (left panel) and by wound-healing migration assay performed for 12 hours (right panel) in the presence or absence of BMP2. Mean ± s.e.m. of three independent experiments (*P<0.001 compared with the GFP-transfected control cells and in the absence of BMP2, One-way ANOVA followed by Bonferroni's multiple comparison test).
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