CENP-A OE contributes to reduced levels of HEC1 and Astrin at kinetochores. (A) Reduced levels of stable end-on attached kinetochores in DLD1^CENP-A cells with overexpressed CENP-A. Images show KT-MT attachment status in DLD1^CENP-A cells with or without DOX treatment. Prior to fixation with ice-cold methanol for 1 min, cells were treated with 10 µM monastrol (3 h) and 10 µM MG132 (90 min), followed by exposure to cold for 10 min. Cells were immunostained with antibodies against NUF2 and β-tubulin and analyzed for kinetochores with end-on (stable and cold resistant) or non–end-on attachment (unstable and cold sensitive) to microtubules. Insets correspond to white-boxed areas in main images. Scale bar: 5 µm (main figures). (B) Reduced levels of HEC1 in DLD1 cells with overexpressed CENP-A. Prism graph for quantification of HEC1 signal intensity at kinetochores in DLD1^CENP-A cells with or without DOX treatment as in Fig. 4 E. (C) Reduced Astrin signal intensity at congressed kinetochores on metaphase plate in DLD1 cells with overexpressed CENP-A. Images show signal intensity of Astrin at kinetochores in DLD1^CENP-A cells with or without DOX treatment. Prior to immunostaining, cells were treated with 10 µM MG132 for 90 min. Cells were immunostained with antibodies against CENP-A and Astrin, and Astrin signal intensity was quantified at congressed kinetochores on metaphase plates. Insets correspond to white-boxed areas in main images. Scale bar: 5 µm (main images), 2 µm (insets). (D) Prism graph shows Astrin signal intensity at kinetochores in DLD1^CENP-A cells with or without DOX treatment. In all prism graphs, each circle represents each kinetochore. “Kts” denotes the number of kinetochore pairs analyzed in number of cells as denoted by “Cells.” Red horizontal lines represent mean signal intensities. Error bars represent SD measured across kinetochores in the indicated number of cells from at least two independent experiments. P values calculated by using the Mann-Whitney U test are indicated.