Figure 4.
CENP-A OE weakens the strength of kinetochores and contributes to reduced stable end-on attachment between kinetochores and microtubules. (A) Reduced interkinetochore distance in DLD1 cells due to weakening of native kinetochores in DLD1 cells overexpressing CENP-A. Images show interkinetochore distance in DLD1CENP-A cells with or without DOX treatment. Prior to immunostaining, cells were treated with 10 µM MG132 for 90 min. Cell were immunostained with antibodies against CENP-A and NUF2, stained with DAPI, and analyzed for interkinetochore distance as determined by the distance between two NUF2 signals in a pair of chromatids. Insets correspond to red-boxed areas in main images. White-boxed areas in insets show examples for kinetochore pairs included in the analysis. Scale bar: 5 µm. (B) Prism graph for quantification of interkinetochore distance in DLD1CENP-A cells with or without DOX treatment. Red horizontal lines represent mean interkinetochore distance. Error bars represent SD across kinetochores measured in the indicated number of cells from three independent experiments. P values were calculated by using the Mann-Whitney U test. (C) Reduced levels of stable end-on attached kinetochores in DLD1 cells overexpressing CENP-A. Images show KT-MT attachments status in DLD1CENP-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 show end-on and non–end-on KT-MT attachment status. Scale bar: 5 µm (main figures). (D) Quantification shows KT-MT attachments status in DLD1CENP-A cells with or without DOX treatment from C. (E) Reduced levels of Astrin-positive kinetochores in DLD1 cells with overexpressed CENP-A. Images show status of Astrin at kinetochores in DLD1CENP-A cells with or without DOX treatment, following the experimental regimen as in C. Cells were immunostained with antibodies against HEC1 and Astrin, stained with DAPI, and analyzed for kinetochores with or without Astrin, which represents end-on versus non–end-on KT-MT attachments, respectively. Insets correspond to white-boxed areas in main images. Scale bar: 5 µm (main images), 0.5 µm (insets). (F) Quantification shows Astrin-positive kinetochores in DLD1CENP-A cells with or without DOX treatment. “Kts” denotes the number of kinetochore pairs analyzed in the indicated number of cells as denoted by “Cells.” Error bars in D and F represent SEM from three independent experiments. P values calculated by using the proportion test (D) and unpaired Student’s t test (F) are indicated.

CENP-A OE weakens the strength of kinetochores and contributes to reduced stable end-on attachment between kinetochores and microtubules. (A) Reduced interkinetochore distance in DLD1 cells due to weakening of native kinetochores in DLD1 cells overexpressing CENP-A. Images show interkinetochore distance in DLD1CENP-A cells with or without DOX treatment. Prior to immunostaining, cells were treated with 10 µM MG132 for 90 min. Cell were immunostained with antibodies against CENP-A and NUF2, stained with DAPI, and analyzed for interkinetochore distance as determined by the distance between two NUF2 signals in a pair of chromatids. Insets correspond to red-boxed areas in main images. White-boxed areas in insets show examples for kinetochore pairs included in the analysis. Scale bar: 5 µm. (B) Prism graph for quantification of interkinetochore distance in DLD1CENP-A cells with or without DOX treatment. Red horizontal lines represent mean interkinetochore distance. Error bars represent SD across kinetochores measured in the indicated number of cells from three independent experiments. P values were calculated by using the Mann-Whitney U test. (C) Reduced levels of stable end-on attached kinetochores in DLD1 cells overexpressing CENP-A. Images show KT-MT attachments status in DLD1CENP-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 show end-on and non–end-on KT-MT attachment status. Scale bar: 5 µm (main figures). (D) Quantification shows KT-MT attachments status in DLD1CENP-A cells with or without DOX treatment from C. (E) Reduced levels of Astrin-positive kinetochores in DLD1 cells with overexpressed CENP-A. Images show status of Astrin at kinetochores in DLD1CENP-A cells with or without DOX treatment, following the experimental regimen as in C. Cells were immunostained with antibodies against HEC1 and Astrin, stained with DAPI, and analyzed for kinetochores with or without Astrin, which represents end-on versus non–end-on KT-MT attachments, respectively. Insets correspond to white-boxed areas in main images. Scale bar: 5 µm (main images), 0.5 µm (insets). (F) Quantification shows Astrin-positive kinetochores in DLD1CENP-A cells with or without DOX treatment. “Kts” denotes the number of kinetochore pairs analyzed in the indicated number of cells as denoted by “Cells.” Error bars in D and F represent SEM from three independent experiments. P values calculated by using the proportion test (D) and unpaired Student’s t test (F) are indicated.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal