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. 2017 May 23;8(29):46781–46800. doi: 10.18632/oncotarget.18108

Figure 6. Reduced levels of CENP-T and Nuf2 at kinetochores contribute to reduced interkinetochore distance in CENP-A overexpressing cells.

Figure 6

A. and C. CENP-T and Nuf2 do not mislocalize in CENP-A overexpressing cells. Representative immunofluorescence images of HeLa FRT/TO mCherry-CENP-A cells untreated or treated with 0.1 μg/ml tetracycline show normal localization of CENP-T (A) and Nuf2 (C), along with CENP-A. Cells were immunostained with indicated antibodies. Scale bar is 5 μm. B. and C. CENP-T and Nuf2 levels are reduced at the centromere and kinetochore, respectively in CENP-A overexpressing cells. Prism graphs show signal intensities of CENP-T at centromeres (B) and Nuf2 at kinetochores D. in metaphase plates of HeLa FRT/TO mCherry-CENP-A cells untreated or treated with 0.1 μg/ml of tetracycline. Each circle represents each centromere (for B) or kinetochore (for D) and ‘Kts’ denotes number of centromeres or kinetochores analyzed in certain number of cells as denoted by ‘cells’. E. Interkinetochore distance is reduced in CENP-A overexpressing cells. Representative immunostained images of HeLa FRT/TO mCherry-CENP-A cells untreated or treated with 0.1 μg/ml of tetracycline show interkinetochore distance. Prior to immunostaining, cells were treated with 10 μM MG132 for 3 hours. Cells were then immunostained with antibodies against CENP-A, Nuf2 as outer kinetochore marker, CREST antisera for centromeres and stained with DAPI for DNA. Insets correspond to red boxed areas in main images. White boxed areas in insets show examples for kinetochore pairs included in analysis. Scale bar is 5 μm. F. Prism graph shows interkinetochore distance ascertained by distance between two Nuf2 signals within aligned chromosomes in MG132-arrested HeLa FRT/TO mCherry-CENP-A cells untreated or treated with 0.1 μg/ml tetracycline. Each circle represents each kinetochore pair and ‘Kts’ denotes number of kinetochore pairs analyzed in certain number of cells as denoted by ‘cells’. Red horizontal lines represent mean signal intensities (B and D) or mean interkinetochore distance (F) as indicated. Error bars represent standard error of mean (SEM) across kinetochores measured from at least two independent experiments. P-values calculated using the Mann-Whitney U test are indicated.