Figure S2.
Effect of a phosphatase inhibitor on the metaphase Hec1-S55 phosphorylation and Hec1-S69 phosphorylation in nontransformed and cancer cell lines.(A) Specificity of Hec1-S55 phosphorylation signal in HeLa and RPE-1 cells in metaphase. Cells were arrested in metaphase in the presence of MG132, treated with a blocking peptide for anti–phospho-Hec1-S55 antibody, 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). (B) Quantification of phospho-Hec1-S55 signal in HeLa and RPE-1 cells in metaphase treated as in A. Relative intensity of the phosphorylated Hec1-S55 signal, which was calculated by dividing the phosphorylation signal with the Hec1 signal on each kinetochore, is displayed as box and dot plots as in Fig. 2 B. The median of mock-treated RPE-1 cells was set as 1. The data represent a minimum of 256 kinetochores from five cells for each condition. P values were obtained using the Mann–Whitney U test. (C) Specificity of Hec1-S55 phosphorylation signal in RPE-1 cells in metaphase. RPE-1 cells depleted of endogenous Hec1 and expressing EGFP-tagged WT Hec1 (WT) or Hec1-S55A, a phosphorylation site mutant, were arrested in metaphase by 2-h MG132 treatment and 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). (D) Quantification of phospho-Hec1-S55 signal in RPE-1 cells in metaphase treated as in C. Intensity of the phosphorylated Hec1-S55 signal on chromosomes is shown. The average of nontransfected RPE-1 cells was set as 1. Error bars represent SD of a minimum of five cells for each condition. P values were obtained using the Tukey–Kramer multiple comparisons test. (E) Hec1-S55 phosphorylation in RPE-1 cells during metaphase in the presence of Calyculin A. Cells were treated with Calyculin A and 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). Cells arrested in metaphase are shown. Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (F) Quantification of the Hec1-S55 phosphorylation signal in RPE-1 cells during metaphase in the presence of Calyculin A. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in E, which was calculated by dividing the phospho-Hec1-S55 signal with the Hec1 signal on each kinetochore, is displayed as box and dot plots as in Fig. 2 B. The median of Calyculin A–treated cells without Aurora inhibitors was set as 1. The data represent a minimum of 346 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (G) Hec1-S55 phosphorylation in HeLa cells during metaphase in the presence of Calyculin A. Cells were treated as in E, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Cells arrested in metaphase are shown. Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (H) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells during metaphase in the presence of Calyculin A. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in G, which was calculated as in F, is displayed as in Fig. 2 B. The median of Calyculin A–treated cells without Aurora inhibitors was set as 1. The data represent a minimum of 265 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (I) Hec1-S69 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-S69 (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). (J) Quantification of the Hec1-S69 phosphorylation signal in HeLa and RPE-1 cells during prometaphase and metaphase. Relative intensity of the phosphorylated Hec1-S69 signal was calculated by dividing the phosphorylation signal by the Hec1 signal on each kinetochore and displayed as in Fig. 2 B. The median of prometaphase in each cell line was set as 1. The data represent a minimum of 194 kinetochores from five cells for each condition. P values were obtained using the Mann–Whitney U test. (K) Hec1-S69 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-S69 (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). (L) Quantification of the Hec1-S69 phosphorylation signal in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Relative intensity of phospho-Hec1-S69 signal in cells treated as in K was calculated and displayed as box and dot plots as in Fig. 2 B. The median of RPE-1 cells was set as 1. The data represent a minimum of 251 kinetochores from five cells for each cell line. P values were obtained using the Steel–Dwass multiple comparisons test. Hec1si, treatment with an siRNA for Hec1.

Effect of a phosphatase inhibitor on the metaphase Hec1-S55 phosphorylation and Hec1-S69 phosphorylation in nontransformed and cancer cell lines. (A) Specificity of Hec1-S55 phosphorylation signal in HeLa and RPE-1 cells in metaphase. Cells were arrested in metaphase in the presence of MG132, treated with a blocking peptide for anti–phospho-Hec1-S55 antibody, 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). (B) Quantification of phospho-Hec1-S55 signal in HeLa and RPE-1 cells in metaphase treated as in A. Relative intensity of the phosphorylated Hec1-S55 signal, which was calculated by dividing the phosphorylation signal with the Hec1 signal on each kinetochore, is displayed as box and dot plots as in Fig. 2 B. The median of mock-treated RPE-1 cells was set as 1. The data represent a minimum of 256 kinetochores from five cells for each condition. P values were obtained using the Mann–Whitney U test. (C) Specificity of Hec1-S55 phosphorylation signal in RPE-1 cells in metaphase. RPE-1 cells depleted of endogenous Hec1 and expressing EGFP-tagged WT Hec1 (WT) or Hec1-S55A, a phosphorylation site mutant, were arrested in metaphase by 2-h MG132 treatment and 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). (D) Quantification of phospho-Hec1-S55 signal in RPE-1 cells in metaphase treated as in C. Intensity of the phosphorylated Hec1-S55 signal on chromosomes is shown. The average of nontransfected RPE-1 cells was set as 1. Error bars represent SD of a minimum of five cells for each condition. P values were obtained using the Tukey–Kramer multiple comparisons test. (E) Hec1-S55 phosphorylation in RPE-1 cells during metaphase in the presence of Calyculin A. Cells were treated with Calyculin A and 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). Cells arrested in metaphase are shown. Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (F) Quantification of the Hec1-S55 phosphorylation signal in RPE-1 cells during metaphase in the presence of Calyculin A. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in E, which was calculated by dividing the phospho-Hec1-S55 signal with the Hec1 signal on each kinetochore, is displayed as box and dot plots as in Fig. 2 B. The median of Calyculin A–treated cells without Aurora inhibitors was set as 1. The data represent a minimum of 346 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (G) Hec1-S55 phosphorylation in HeLa cells during metaphase in the presence of Calyculin A. Cells were treated as in E, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Cells arrested in metaphase are shown. Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (H) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells during metaphase in the presence of Calyculin A. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in G, which was calculated as in F, is displayed as in Fig. 2 B. The median of Calyculin A–treated cells without Aurora inhibitors was set as 1. The data represent a minimum of 265 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (I) Hec1-S69 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-S69 (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). (J) Quantification of the Hec1-S69 phosphorylation signal in HeLa and RPE-1 cells during prometaphase and metaphase. Relative intensity of the phosphorylated Hec1-S69 signal was calculated by dividing the phosphorylation signal by the Hec1 signal on each kinetochore and displayed as in Fig. 2 B. The median of prometaphase in each cell line was set as 1. The data represent a minimum of 194 kinetochores from five cells for each condition. P values were obtained using the Mann–Whitney U test. (K) Hec1-S69 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-S69 (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). (L) Quantification of the Hec1-S69 phosphorylation signal in nontransformed (blue), non-CIN (purple), and CIN (red) cancer cell lines during metaphase. Relative intensity of phospho-Hec1-S69 signal in cells treated as in K was calculated and displayed as box and dot plots as in Fig. 2 B. The median of RPE-1 cells was set as 1. The data represent a minimum of 251 kinetochores from five cells for each cell line. P values were obtained using the Steel–Dwass multiple comparisons test. Hec1si, treatment with an siRNA for Hec1.

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