Effects of vimentin repositioning on the microtubule cytoskeleton. (A and B) COS-7 cells were co-transfected with Vim-mCh-FKBP and either FRB-BFP-GCN4-ppKin14 (A) or HA-KIF5A-FRB (B), with non-transfected cells as controls. Transfected cells are outlined with a dashed line. After 1 h of treatment with or without rapalog, cells were fixed for analysis. Representative fluorescent images display the microtubule network, labeled with antibodies against tyrosinated α-tubulin (Tyr-tubulin). Microtubules are further color-coded to indicate radial (cyan) and non-radial (yellow) orientations. (C) Quantification of tyrosinated α-tubulin intensity in cells expressing Vim-mCh-FKBP along with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB, normalized to non-transfected cells, with or without rapalog treatment. n = 21–23 cells analyzed across two independent experiments. (D) Quantification of the ratio of non-radial to radial tyrosinated microtubules in non-transfected cells and cells expressing Vim-mCh-FKBP with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB, with or without rapalog treatment. n = 20–22 cells were analyzed across two independent experiments. (E and F) Representative images of U2OS cells stained for acetylated microtubules (Ac-tubulin), showing either untransfected cells or cells co-expressing Vim-mCh-FKBP with either FRB-BFP-GCN4-ppKin14 (E) or HA-KIF5A-FRB (F). Transfected cells are outlined with a dashed line. After 1 h of treatment with or without rapalog, cells were fixed for analysis. (G) Quantification of normalized acetylated microtubule intensity in untransfected U2OS cells and cells co-expressing Vim-mCh-FKBP with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB, in the presence and absence of rapalog. n = 37–49 cells were analyzed across three independent experiments. Plots indicate mean ± SD, with individual cell measurements shown as dots. ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 as assessed by Kruskal–Wallis test.