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Fig. 2. BMP2 induces actin cytoskeleton reorganization. (A) C2C12 cells were stimulated with 3 nM BMP2 for the indicated times. Actin was visualized with TRITC-conjugated phalloidin using a confocal microscope. Arrows indicate actin cortical protrusions. Scale bar: 20 µm. (B) Serum-starved C2C12 cells were pretreated with 2 µM cytochalasin D (Cyto D) for 20 minutes and allowed to recover in the absence or presence of 3 nM BMP2 for 1 hour. Cell lysates were analyzed by immunoblotting with anti-phospho-Smad1 and reprobed with anti-
-tubulin antibodies. (C) Cells treated as above were allowed to recover in the absence or presence of 3 nM BMP2. Upper row shows cells before and after 2 µM Cyto D treatment. Middle and lower rows show recovery in the absence or presence of BMP2, respectively. Scale bar: 20 µm. A quantitative analysis of the formation of stress fibers in the absence of BMP2 or cells showing cortical actin protrusions as a percent of total in the presence of BMP2 are shown. Data are mean ± s.e.m. of at least 200 cells obtained in different fields from five independent experiments. (D) Serum-starved C2C12 cells (Control) were pre-treated with 2 µM cytochalasin D (Cyto D) for 20 minutes and after washing, were allowed to recover in the presence of 3 nM BMP2 for different times. Actin was visualized with TRITC-conjugated phalloidin using a confocal microscope. Arrows indicate actin cortical protrusions that were evident within the first 60 minutes and cells then recovered their initial aspect showing abundant stress fibers. Scale bar: 20 µm.
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