Reduced localization of proteins at kinetochore in cells with CENP-A OE. (A and B) Reduced CENP-T signal intensities in DLD1 cells with overexpressed and mislocalized CENP-A. Images (A) of CENP-T localization at centromeres on metaphase plates, and prism graph (B) shows CENP-T signal intensity at centromeres in DLD1^CENP-A cells with or without DOX treatment. (C and D) Reduced NUF2 signal intensities in DLD1 cells with overexpressed and mislocalized CENP-A. Images (C) of NUF2 localization at kinetochores on metaphase plates, and prism graph (D) shows NUF2 signal intensity at kinetochores in DLD1^CENP-A cells with or without DOX treatment. (E and F) Reduced levels of Mis12 at centromeres in DLD1 cells with overexpressed CENP-A. Images (E) of localization of Mis12 at kinetochores on metaphase plates, and prism graph (D) shows Mis12 signal intensity at kinetochores in DLD1^CENP-A cells with or without DOX treatment. For all experiments in this figure, before immunostaining, cells were treated with 10 µM MG132 for 90 min, followed by fixation with ice-cold methanol for 1 min, and then immunostaining with antibodies as indicated. Cells were stained with DAPI and analyzed for signal intensities at kinetochores on metaphase plates. For all prism graphs in B, D, and F, each circle represents each kinetochore pair and “Kts” denotes the number of kinetochore pairs analyzed in a certain number of cells as denoted by “Cells.” Red horizontal lines represent mean signal intensities as indicated. Error bars represent SD measured across kinetochores in the indicated number of cells from three independent experiments. P values were calculated by using the Mann-Whitney U test. Scale bar: 5 µm.