Fig. 6. Capn4^–/– fibroblasts are defective in adhesiveness and adhesion: stress fiber linkage. (A) Rescued Capn4^–/– fibroblasts, Capn1- and Capn2-knockdown and calpastatin-overexpressing MEF cells, but not Capn4^–/– cells, adhere strongly to the substrate in a centrifugation assay. Each bar represents mean ± s.e.m. results from three separate experiments, expressed as a percentage of control as defined by wild-type fibroblasts. Actin and vinculin immunofluorescence of representative control fibroblasts (B,C) and Capn4^–/– fibroblasts (D,E) show the abnormal organization of these structures in the knockout cells. Scale bar: 10 µm. (F) Colocalization analysis of actin and vinculin indicates a decreased association of prominent actin fibers with vinculin-containing adhesions in the Capn4^–/– fibroblasts compared with the control and Capn1- and Capn2-silenced fibroblasts, as well as cells overexpressing calpastatin. The analysis involves counting the total number of vinculin-containing adhesions and those associated with actin stress fibers in the anterior region (n=15 cells).