Figure 1.
Overexpression of CENP-A contributes to mislocalization of CENP-A and CENP-C to non-centromeric regions and chromosome missegregation in DLD1 cells. (A) Inducible OE of YFP–CENP-A leads to its mislocalization to chromosome arms in DLD1 cells. Chromosome spread images showing localization of CENP-A to centromeres in DLD1CENP-A cells without DOX treatment and to chromosome arms in cells with DOX treatment. Following preparation of chromosome spreads, cells were fixed in 4% PFA, followed by trypsinization with 0.1% Triton X-100 in PBS. Cells were immunostained with antibody against CENP-A and stained with DAPI. Scale bar: 2 µm. (B) Prism graphs for quantification of CENP-A signal intensities at non-centromeric (left) and centromeric (right) regions in chromosome spreads of DLD1CENP-A cells from A. Each circle represents a spot on a chromosome. “Cells” and “chr” denote number of cells and chromosomes analyzed, respectively. Red horizontal lines represent mean signal intensity as indicated. Error bars represent SD across areas measured in number of cells from three independent experiments. P values were calculated by using the Mann-Whitney U test. (C) Reduced levels of CENP-C at centromeres with mislocalization to chromosome arms in DLD1 cells overexpressing CENP-A. Chromosome spread images show localization of CENP-C in DLD1CENP-A cells with or without DOX treatment, followed by immunostaining with antibodies against CENP-A and CENP-C, staining with DAPI, and analysis for CENP-C signal intensity at centromeric and non-centromeric regions. Scale bar: 2 µm. (D) Prism graphs for quantification of CENP-C signal intensities at non-centromeric (left) and centromeric (right) regions in chromosome spreads of DLD1CENP-A cells treated as in C. Each circle represents a spot on a chromosome. “Cells” and “chr” denote number of cells and chromosomes analyzed, respectively. Red horizontal lines represent mean signal intensity as indicated. Error bars represent SD across areas measured in number of cells from two independent experiments. P values were calculated by using the Mann-Whitney U test. (E) Increased chromosome segregation defects in DLD1 cells with overexpressed and mislocalized CENP-A. Images show chromosome segregation status in DLD1CENP-A cells with or without DOX treatment. Cells were immunostained with antibodies against CENP-A, stained with DAPI, and analyzed for chromosome segregation status. Yellow arrow shows missegregated chromosomes in DOX-treated DLD1CENP-A cells. Scale bar: 5 µm. (F) Quantification shows the proportion of cells with defective chromosome segregation in DLD1CENP-A cells treated as in E. Error bars represent SEM from three independent experiments. P values were calculated by using the unpaired Student’s t test.

Overexpression of CENP-A contributes to mislocalization of CENP-A and CENP-C to non-centromeric regions and chromosome missegregation in DLD1 cells. (A) Inducible OE of YFP–CENP-A leads to its mislocalization to chromosome arms in DLD1 cells. Chromosome spread images showing localization of CENP-A to centromeres in DLD1CENP-A cells without DOX treatment and to chromosome arms in cells with DOX treatment. Following preparation of chromosome spreads, cells were fixed in 4% PFA, followed by trypsinization with 0.1% Triton X-100 in PBS. Cells were immunostained with antibody against CENP-A and stained with DAPI. Scale bar: 2 µm. (B) Prism graphs for quantification of CENP-A signal intensities at non-centromeric (left) and centromeric (right) regions in chromosome spreads of DLD1CENP-A cells from A. Each circle represents a spot on a chromosome. “Cells” and “chr” denote number of cells and chromosomes analyzed, respectively. Red horizontal lines represent mean signal intensity as indicated. Error bars represent SD across areas measured in number of cells from three independent experiments. P values were calculated by using the Mann-Whitney U test. (C) Reduced levels of CENP-C at centromeres with mislocalization to chromosome arms in DLD1 cells overexpressing CENP-A. Chromosome spread images show localization of CENP-C in DLD1CENP-A cells with or without DOX treatment, followed by immunostaining with antibodies against CENP-A and CENP-C, staining with DAPI, and analysis for CENP-C signal intensity at centromeric and non-centromeric regions. Scale bar: 2 µm. (D) Prism graphs for quantification of CENP-C signal intensities at non-centromeric (left) and centromeric (right) regions in chromosome spreads of DLD1CENP-A cells treated as in C. Each circle represents a spot on a chromosome. “Cells” and “chr” denote number of cells and chromosomes analyzed, respectively. Red horizontal lines represent mean signal intensity as indicated. Error bars represent SD across areas measured in number of cells from two independent experiments. P values were calculated by using the Mann-Whitney U test. (E) Increased chromosome segregation defects in DLD1 cells with overexpressed and mislocalized CENP-A. Images show chromosome segregation status in DLD1CENP-A cells with or without DOX treatment. Cells were immunostained with antibodies against CENP-A, stained with DAPI, and analyzed for chromosome segregation status. Yellow arrow shows missegregated chromosomes in DOX-treated DLD1CENP-A cells. Scale bar: 5 µm. (F) Quantification shows the proportion of cells with defective chromosome segregation in DLD1CENP-A cells treated as in E. Error bars represent SEM from three independent experiments. P values were calculated by using the unpaired Student’s t test.

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