Fig. 2. Quantification of the Smad7-dependent activation of the actin filament system. (A) Confocal image of a representative PC-3U/pMEP4-S7 cell treated with 1 µM CdCl₂ for 12 hours. Flag-Smad7 and filamentous actin was detected as in Fig. 1. The fluorescence intensity changes across the yellow line in the direction of the arrowhead were plotted as the line intensities in the histogram in the lower panel. The red channel (Ch1-T1) represents filamentous actin and the green channel (Ch2-T2) represents Flag-Smad7. (B) Quantification of the effect of CdCl₂ and TGF-ß treatment of the PC-3U/pMEP4-S7 cells in Fig. 1 on membrane ruffling and stress-fiber formation. At least 300 cells were counted under the microscope for each condition. (C) PC-3U/pMEP4-S7 cells were serum-starved in 1% FBS for 12 hours and stimulated with 5 ng/ml TGF-ß for 30 minutes, 12 hours and 24 hours to show the effect on the actin filament system in the absence of CdCl₂-induced Smad7 expression. Parental PC-3U cells were mock-treated with 1 µM CdCl₂ for 12 hours or stimulated with 5 nM TGF-ß for 12 hours to show that CdCl₂ alone did not induce any effect on the actin filament system. Bars, 20 µm.