Fig. 4. Force increases actin in collagen-bead-associated attachment complexes. (A) Bead-associated proteins from Rat-2 cells treated with force. Cells were incubated with collagen-coated beads. Cells were lysed in cytoskeletal stabilizing buffer containing phalloidin (1 µM). Soluble material was immunoblotted for GAPDH. Beads were removed magnetically and counted. Proteins from equal numbers of beads were immunoblotted for β-actin. (B) The relative change of bead-associated actin was computed from densitometry measurements of immunoblots. Data are given as the mean ± s.d. compared with the no-force ratio (designated as 1). Force increased the amount of actin filaments 2.5-fold above controls at 15 minutes (P<0.01). (C) Rhodamine-phalloidin staining of cells not subjected to force (NF) or subjected to force for 15 minutes (F15'). Notice the staining around beads in force-treated sample. (D) Fluorescence intensity around collagen beads was quantified in 4-µm circular regions of interest around beads with a CCD camera. The results are given as the mean ± s.d. fluorescence units and are derived from the counts of beads in 100 cells for each treatment in each experiment. (E) Cells were transiently transfected with vehicle (V), siRNA targeting cofilin (Co) or siRNA targeting GFP (G) for 1, 2 or 3 days. Immunoblots show that siRNA targeting cofilin suppresses cofilin protein by >80% after 2 days transfection.