Fig 1: Farnesylation promotes interaction of CENP-E with fibrous corona proteins. (A) Immunostaining of ZWILCH (magenta) in RPE cells that overexpress LAP-CENP-E FL or LAP-CENP-E 1301-C. Bar, 5 μm. (B) Immunostaining of ZWILCH (magenta) in control and 5uM lonafarnib-treated HeLa cells that overexpress LAP-CENP-E 2111-C. Bar, 5 μm. (C) Immunostaining of ZWILCH (magenta) in DMSO/lonafarnib-treated RPE cells that overexpress LAP-CENP-E 2111-C and LAP-CENP-E 2111-C C2698A in the presence of nocodazole treatment. Bar, 5 μm; inset bar, 1 μm. (D) Volcano plot shows top enriched proteins bound to the streptavidin beads incubation with extracts of HeLa cells expressing LAP-TurboID-CENP-E 2111-C C-10A9 (left) or LAP-TurboID-CENP-E 2111-2689 (right). (E) Volcano plot shows top enriched proteins bound to the streptavidin beads incubation with extracts of HeLa cells expressing LAP-TurboID-CENP-E 2111-C C-10A7 (left) or LAP-TurboID-CENP-E 2111-C C2698A (right). (F) Immunostaining of BUBR1 (green)/ZWINT-1 (green), CENP-E (magenta), and CENP-C (blue) in RPE1 cells treated with STLC and GSK923295 overnight. Bar, 5 μm; inset bar, 1 μm. (G) Immunostaining of SPINDLY (green), CENP-E (magenta), and CENP-C (blue) in RPE1 cells treated with STLC and GSK923295 overnight in the presence of DMSO or 5 μM lonafarnib. Bar, 5 μm. (H) Relative intensity of SPINDLY at the spindle pole in RPE1 cells treated with STLC and GSK923295 overnight in the presence of DMSO or 5 μM lonafarnib. n > 60 cells were quantified for each condition. Experiments were performed in triplicate and data from each trial was color-coded. Average value of each trial is given by a filled triangle of the corresponding color. Results are represented as mean ± SD; error bars represent SD. The P values represent a paired two-sample two-tailed t test. (I) Relative intensity of ROD at the spindle pole in RPE1 cells treated with STLC and GSK923295 overnight in the presence of DMSO or 5 μM lonafarnib. n > 60 cells were quantified for each condition. Experiments were performed in triplicate and data from each trial was color-coded. Average value of each trial is given by a filled triangle of the corresponding color. Results are represented as mean ± SD; error bars represent SD. The P values represent a paired two-sample two-tailed t test. (J) Relative intensity of MAD1 at the spindle pole in RPE1 cells treated with STLC and GSK923295 overnight in the presence of DMSO or 5 μM lonafarnib. n > 60 cells were quantified for each condition. Experiments were performed in triplicate and data from each trial was color-coded. Average value of each trial is given by a filled triangle of the corresponding color. Results are represented as mean ± SD; error bars represent SD. The P values represent a paired two-sample two-tailed t test. (K) Immunostaining of ZWILCH (magenta), CENP-E (gray), and CENP-C (blue) in control and CENP-E–depleted HeLa cells and CENP-E–depleted HeLa cells that overexpress the indicated LAP-tagged CENP-E mutant after STLC and GSK923295 treatment overnight. Bar, 5 μm. LAP-tagged CENP-E mutants were created by fusing the N-terminal 1–426 (comprising the motor domain) to the C-terminal (KT-binding domain and MT-binding domain). Rabbit polyclonal CENP-E antibody recognizes human CENP-E 955–1571. (L) Relative intensity of ZWILCH at the spindle pole in control and CENP-E–depleted HeLa cells and CENP-E–depleted HeLa cells that inducibly overexpress the indicated LAP-tagged CENP-E mutant after STLC and GSK923295 treatment overnight. n > 60 cells were quantified for each condition. Experiments were performed in triplicate and data from each trial was color-coded. Average value of each trial is given by a filled triangle of the corresponding color. Results are represented as mean ± SD; error bars represent SD. The P values represent a paired two-sample two-tailed t test.