Effects of vimentin repositioning on the actin cytoskeleton, cell spreading, and focal adhesions. (A–F) COS-7 (A–C) or U2OS (D–F) cells were co-transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14, while non-transfected cells served as controls. Transfected cells are outlined with a dashed line. (A and D) After 1 h of treatment with or without rapalog, the cells were fixed and stained for total vimentin, paxillin, and actin using phalloidin. (B and E) Quantification of the total cell area based on the phalloidin staining in transfected (T) and untransfected (U) cells, with and without rapalog treatment. Dashed boxes show regions enlarged in the zoom panels. (C and F) Quantification of the total focal adhesion number based on paxillin staining, normalized to cell area as determined by phalloidin staining in both transfected and untransfected cells, with or without rapalog treatment. Measurements were collected from n = 28–31 cells in B and C and from n = 27–29 cells in E and F across three independent experiments. The plots display the mean ± SD, with individual cell measurements represented as dots. ns, not significant, determined by Kruskal–Wallis analysis.