Fig. 4. Effects of Smad7 depletion on actin organization and activation of Rho GTPases. (A) PC-3U/AS-S7 cells (upper panel) or parental PC-3U cells (lower panel) were serum-starved in 1% FBS for 12 hours and treated with 5 ng/ml TGF-ß for 15 minutes, 30 minutes, 12 hours and 24 hours. Actin filaments were visualized by TRITC-labeled phalloidin. Arrowheads in the lower panel indicate parental cells with membrane ruffles, a response absent in PC-3U/AS-S7 cells. Bar, 20 µm. (B) Quantification of the effect of TGF-ß treatment of the PC-3U/AS-S7 and PC-3U cells in panel A on membrane ruffling and stress-fiber formation. At least 300 cells were counted under the microscope for each condition. (C) PC-3U/AS-S7 cells were serum-starved in 1% FBS for 12 hours and treated with 5 ng/ml TGF-ß for 5 minutes to 48 hours. As a positive control, PC-3U cells were stimulated with 5 ng/ml of TGF-ß for 15 minutes and 12 hours. The amount of active, GTP-loaded Rac1, Cdc42 and RhoA was determined by GST pull-down assays with GST-PAK-CRIB, GST-WASP-CRIB, or GST-Rhotekin, respectively. Rac1, Cdc42 and RhoA were detected by immunoblotting using antibodies specific for the respective GTPase. (D) Immunoblots were analyzed by densitometry and the data are combined into diagrams showing the activation of Rac1, Cdc42, and RhoA. Each column represents the mean + s.e.m. from three independent experiments.