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Fig. 10. Role of BMPRII in BMP2-dependent actin cytoskeleton reorganization and cell migration. (A) Chemotaxis assay. Serum-starved C2C12 cells were allowed to migrate for 2 hours in the presence or absence of 30 pM BMP7 as described in Materials and Methods. Quantitative analysis of eight random fields from three independent experiments (Mean ± s.e.m.; *P<0.0001, paired t-test). (B) Wound-healing migration assay. Serum-starved wounded C2C12 cell monolayers were allowed to migrate in the presence or absence of 3 nM BMP2 or 3 nM BMP7 for 24 hours. The graph indicates the invaded area. Mean ± s.e.m. of three independent experiments (*P<0.001, paired t-test). (C) Immunoblot analysis of BMPRII expression in C2C12 cells transfected with the indicated siRNA for 48 hours. (D) C2C12 cells were transfected with the indicated siRNA for 48 hours and serum-starved for 16 hours. Then, cells were pretreated with 2 µM cytochalasin D for 20 minutes and allowed to recover in the absence or presence of 3 nM BMP2 for 1 hour. Merged images of phalloidin and GFP signals 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<0.05, paired t-test compared with the condition in the absence of BMP2). (E) C2C12 cells were transfected with the indicated siRNA for 48 hours and serum-starved for 16 hours. Cell monolayers were wounded and allowed to migrate for 24 hours in the presence or absence of 3 nM BMP2. The graph indicates the invaded area. Mean ± s.e.m. from three independent experiments (*P<0.001 compared with the condition in the absence of BMP2, one-way ANOVA followed by Bonferroni's multiple comparison test).
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