Figure 2.
Hec1-S55 is phosphorylated during metaphase by Aurora A, which is reduced in cancer cell lines.(A) Hec1-S55 phosphorylation in HeLa and RPE-1 cells during prometaphase (Prometa) and metaphase (Meta). Cells were fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bars: 5 µm (white), 500 nm (yellow). (B) Quantification of phospho–Hec1-S55 signal in HeLa and RPE-1 cells during prometaphase and metaphase. Relative intensity of the phosphorylated Hec1-S55 signal in cells treated as in A, which was calculated by dividing the phosphorylation signal with Hec1 signal on each kinetochore, 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. The median of prometaphase in each cell line was set as 1. The data represent a minimum of 394 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. (C) Hec1-S55 phosphorylation during metaphase is dependent on Aurora A. Cells were treated with either DMSO or AZD1152 and/or MLN8237 for the last 1 h of the 3-h MG132 treatment, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bars: 5 µm (white), 500 nm (yellow). (D) Quantification of the Hec1-S55 phosphorylation signal in HeLa and RPE-1 cells during metaphase. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in C, which was calculated by dividing phospho-Hec1-S55 signal with Hec1 signal on each kinetochore, displayed as box and dot plots as in B. The median of DMSO-treated RPE-1 cells was set as 1. The data represent a minimum of 294 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (E) Hec1-S55 phosphorylation in nontransformed and cancer cell lines during metaphase. Cells were treated with MG132 for 3 h and then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (F) Quantification of Hec1-S55 phosphorylation signal in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Relative intensity of phospho-Hec1-S55 signal in cells treated as in E was calculated and displayed as box and dot plots as in B. The median of RPE-1 cells was set as 1. The data represent a minimum of 246 kinetochores from five cells for each cell line. P values were obtained using the Steel multiple comparisons test.

Hec1-S55 is phosphorylated during metaphase by Aurora A, which is reduced in cancer cell lines. (A) Hec1-S55 phosphorylation in HeLa and RPE-1 cells during prometaphase (Prometa) and metaphase (Meta). Cells were fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bars: 5 µm (white), 500 nm (yellow). (B) Quantification of phospho–Hec1-S55 signal in HeLa and RPE-1 cells during prometaphase and metaphase. Relative intensity of the phosphorylated Hec1-S55 signal in cells treated as in A, which was calculated by dividing the phosphorylation signal with Hec1 signal on each kinetochore, 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. The median of prometaphase in each cell line was set as 1. The data represent a minimum of 394 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. (C) Hec1-S55 phosphorylation during metaphase is dependent on Aurora A. Cells were treated with either DMSO or AZD1152 and/or MLN8237 for the last 1 h of the 3-h MG132 treatment, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bars: 5 µm (white), 500 nm (yellow). (D) Quantification of the Hec1-S55 phosphorylation signal in HeLa and RPE-1 cells during metaphase. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in C, which was calculated by dividing phospho-Hec1-S55 signal with Hec1 signal on each kinetochore, displayed as box and dot plots as in B. The median of DMSO-treated RPE-1 cells was set as 1. The data represent a minimum of 294 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (E) Hec1-S55 phosphorylation in nontransformed and cancer cell lines during metaphase. Cells were treated with MG132 for 3 h and then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (F) Quantification of Hec1-S55 phosphorylation signal in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Relative intensity of phospho-Hec1-S55 signal in cells treated as in E was calculated and displayed as box and dot plots as in B. The median of RPE-1 cells was set as 1. The data represent a minimum of 246 kinetochores from five cells for each cell line. P values were obtained using the Steel multiple comparisons test.

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