Figure S1.
Cancer cell lines show reduced displacement of kinetochores from the spindle equator during metaphase.(A) Trajectories of kinetochores in RPE-1 and HeLa cells during unperturbed mitotic progression through metaphase to anaphase. The blue and red trajectories show the movements of a pair of sister kinetochores plotted as the distance from the spindle equator. (B) DAP measurements of kinetochore position in RPE-1 and HeLa cells during metaphase in the presence of MG132 or early and late metaphase without MG132 treatment. Error bars represent SD of 10 kinetochore pairs from three cells. P values were obtained using the Tukey–Kramer multiple comparisons test. (C) Quantification of cells containing micronuclei in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines. Cells were stained with DAPI after fixation, and interphase cells were observed for the presence of micronuclei (yellow arrowhead in the inset, scale bar: 5 µm). At least 223 cells were observed for each cell line. Error bars represent SD of three independent experiments. P values were obtained using the Tukey–Kramer multiple comparisons test. (D) Quantification of cells showing the number of FISH signals different from the modal number in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines. Cells were fixed and stained with FISH probes for chromosomes (Ch.) 7 and 12 (inset, scale bar: 10 µm). Modal number of FISH signals was determined for each probe in each cell line. At least 165 cells were observed for each cell line. Error bars represent SD of three independent experiments. P values were obtained using the Tukey–Kramer multiple comparisons test. (E) Immunostaining of metaphase cells in nontransformed and cancer cell lines. Cells were treated with MG132 for 3 h and then fixed and stained with anti-Hec1 (green) and anti-pericentrin (PCNT; red) antibodies. DNA was stained with DAPI (blue). Scale bar: 5 µm. (F) Schematic diagram of quantifying variance (V) of kinetochore distance from the spindle equator during metaphase. (G) Quantification of variance of kinetochore distance from the spindle equator in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Cells were treated as in E. The data represent the average variance of 10 cells, in which a minimum of 368 kinetochore pairs were quantified. Error bars represent SD. P values were obtained using the Tukey–Kramer multiple comparisons test. (H) Spindle length in metaphase cells. Cells were treated, fixed, and stained as in E, and spindle length in metaphase cells was measured as distance between pericentrin signals. 10 cells were observed for each cell line. Error bars represent SD. P values were obtained using the Tukey–Kramer multiple comparisons test. (I) Cell size in metaphase cells. Cells were treated, fixed, and stained as in E, and the diameters of metaphase cells, both longitudinal (blue) and parallel (red) to the spindle axes in bright field images, were measured. 10 cells were observed for each cell line. Error bars represent SD. P values were obtained using the Tukey–Kramer multiple comparisons test. (J) Interkinetochore distance in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Cells were treated, fixed, and stained as in E, and interkinetochore distance was measured for a minimum of 102 sister kinetochore (KT) pairs from 10 cells for each cell line, displayed as box and dot plots. The bottom and top of the box show the lower and upper quartile values, respectively. The median is indicated with a bar in the box, and the whiskers denote the range within 1.5× size of the box. P values were obtained using the Steel–Dwass multiple comparisons test.

Cancer cell lines show reduced displacement of kinetochores from the spindle equator during metaphase. (A) Trajectories of kinetochores in RPE-1 and HeLa cells during unperturbed mitotic progression through metaphase to anaphase. The blue and red trajectories show the movements of a pair of sister kinetochores plotted as the distance from the spindle equator. (B) DAP measurements of kinetochore position in RPE-1 and HeLa cells during metaphase in the presence of MG132 or early and late metaphase without MG132 treatment. Error bars represent SD of 10 kinetochore pairs from three cells. P values were obtained using the Tukey–Kramer multiple comparisons test. (C) Quantification of cells containing micronuclei in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines. Cells were stained with DAPI after fixation, and interphase cells were observed for the presence of micronuclei (yellow arrowhead in the inset, scale bar: 5 µm). At least 223 cells were observed for each cell line. Error bars represent SD of three independent experiments. P values were obtained using the Tukey–Kramer multiple comparisons test. (D) Quantification of cells showing the number of FISH signals different from the modal number in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines. Cells were fixed and stained with FISH probes for chromosomes (Ch.) 7 and 12 (inset, scale bar: 10 µm). Modal number of FISH signals was determined for each probe in each cell line. At least 165 cells were observed for each cell line. Error bars represent SD of three independent experiments. P values were obtained using the Tukey–Kramer multiple comparisons test. (E) Immunostaining of metaphase cells in nontransformed and cancer cell lines. Cells were treated with MG132 for 3 h and then fixed and stained with anti-Hec1 (green) and anti-pericentrin (PCNT; red) antibodies. DNA was stained with DAPI (blue). Scale bar: 5 µm. (F) Schematic diagram of quantifying variance (V) of kinetochore distance from the spindle equator during metaphase. (G) Quantification of variance of kinetochore distance from the spindle equator in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Cells were treated as in E. The data represent the average variance of 10 cells, in which a minimum of 368 kinetochore pairs were quantified. Error bars represent SD. P values were obtained using the Tukey–Kramer multiple comparisons test. (H) Spindle length in metaphase cells. Cells were treated, fixed, and stained as in E, and spindle length in metaphase cells was measured as distance between pericentrin signals. 10 cells were observed for each cell line. Error bars represent SD. P values were obtained using the Tukey–Kramer multiple comparisons test. (I) Cell size in metaphase cells. Cells were treated, fixed, and stained as in E, and the diameters of metaphase cells, both longitudinal (blue) and parallel (red) to the spindle axes in bright field images, were measured. 10 cells were observed for each cell line. Error bars represent SD. P values were obtained using the Tukey–Kramer multiple comparisons test. (J) Interkinetochore distance in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Cells were treated, fixed, and stained as in E, and interkinetochore distance was measured for a minimum of 102 sister kinetochore (KT) pairs from 10 cells for each cell line, displayed as box and dot plots. The bottom and top of the box show the lower and upper quartile values, respectively. The median is indicated with a bar in the box, and the whiskers denote the range within 1.5× size of the box. P values were obtained using the Steel–Dwass multiple comparisons test.

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