Figure S5.
Chromosome oscillation and the Aurora A–dependent Hec1 phosphorylation promote each other and facilitate the correction of erroneous kinetochore–microtubule attachments during metaphase.(A) Efficiency of Kif18A RNAi. Lysate of cells transfected with an siRNA against Kif18A was subjected to immunoblot analysis using antibodies as indicated. (B) Trajectories of kinetochores in HeLa cells arrested in metaphase in the presence or absence of BTB-1, a Kif18A inhibitor. Cells were treated with or without BTB-1 for the last 1 h of a 2-h MG132 treatment and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with BTB-1 plotted as the distance from the spindle equator. See also Video 4. (C) DAP measurements of kinetochore position in HeLa cells treated as in B. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (D) Hec1-S55 phosphorylation in HeLa cells during metaphase in the presence of BTB-1. Cells were treated with BTB-1 and either DMSO or AZD1152 and/or MLN8237 for the last 1 h of a 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). (E) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells treated as in D, which was calculated and displayed as box and dot plots as in Fig. 2 B. The median of cells treated with BTB-1 without Aurora inhibitors was set as 1. The data represent a minimum of 347 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. (F) Trajectories of kinetochores in HeLa cells arrested in metaphase in the presence or absence of Calyculin A. Cells were treated with or without Calyculin A for the last 1 h of a 2-h MG132 treatment, then observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with Calyculin A, plotted as the distance from the spindle equator. See also Video 5. (G) DAP measurements of kinetochore position in RPE-1 cells treated as in F. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (H) Hec1-S69 phosphorylation in HeLa cells during metaphase in the presence of BTB-1 or UMK57, a MCAK potentiator. Cells were treated with BTB-1 or UMK57 for the last 1 h of a 3-h MG132 treatment, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S69 (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). (I) Quantification of the Hec1-S69 phosphorylation signal in HeLa cells treated as in H, which was calculated and displayed as in Fig. 2 B. The median of DMSO-treated cells was set as 1. The data represent a minimum of 259 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (J) Expression of Hec1 constructs in RPE-1 cells depleted of endogenous Hec1. Lysate of Hec1-depleted cells transfected with each Hec1 construct was subjected to immunoblot analysis using antibodies as indicated. (K) Kinetochore–microtubule attachments in metaphase HeLa cells. HeLa cells expressing EGFP–CENP-A were subjected to immunostaining with an antibody against GFP (green) and α-tubulin (red). DNA was stained with DAPI (blue). Magnified view of kinetochore–microtubule attachments in selected Z-slices are shown in insets, where merotelic attachment was judged by the presence of microtubules between sister kinetochores. Scale bar: 5 µm (white), 500 nm (yellow). (L) Quantification of the kinetochore pairs forming merotelic attachment in HeLa cells arrested in metaphase in the presence of BTB-1 or UMK57. HeLa cells expressing EGFP–CENP-A were fixed and stained as in K. The data represent the percentage of merotelic attachment, and error bars represent SD of three independent experiments in which a minimum of 100 kinetochore–microtubule attachments in a minimum of 10 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (M) Quantification of the kinetochore pairs in RPE-1 cells arrested in metaphase forming merotelic attachment in the presence of AZD1152 or MLN8237. RPE-1 cells expressing EGFP–CENP-A were fixed and stained as in K. The data represent the percentage of merotelic attachment, and error bars represent SD of three independent experiments in which a minimum of 100 kinetochore–microtubule attachments in a minimum of 10 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (N) Spindle morphology of RPE-1 cells treated with or without Aurora inhibitors. Cells were treated with either DMSO or AZD1152 and/or MLN8237 for the last 1 h of the 3-h MG132 treatment, then incubated on ice for 0 or 10 min before fixation and stained with an anti–α-tubulin antibody (α-tub; green). DNA was stained with DAPI (blue). Scale bars: 5 µm. (O) Quantification of microtubule signals on the spindle. The α-tubulin signal intensities on the spindle of cells treated as in N were measured. The intensity of DMSO-treated cells without cold treatment 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. (P) Expression of Aurora A and TPX2 on the spindle in nontransformed and cancer cell lines during metaphase. Cells were treated with MG132 for 3 h and then fixed and stained with anti–Aurora A (AurA; red) and anti-TPX2 (green) antibodies. DNA was stained with DAPI (blue). Scale bar: 5 µm. Signal intensity of Aurora A and TPX2 and the signal ratio of Aurora A to TPX2 are shown in the right graphs. The average values in RPE-1 cells were set as 1. Error bars represent SD of five independent experiments in which a minimum of 10 cells were observed for each cell line. P value was obtained using the Tukey-Kramer multiple comparisons test.

Chromosome oscillation and the Aurora A–dependent Hec1 phosphorylation promote each other and facilitate the correction of erroneous kinetochore–microtubule attachments during metaphase. (A) Efficiency of Kif18A RNAi. Lysate of cells transfected with an siRNA against Kif18A was subjected to immunoblot analysis using antibodies as indicated. (B) Trajectories of kinetochores in HeLa cells arrested in metaphase in the presence or absence of BTB-1, a Kif18A inhibitor. Cells were treated with or without BTB-1 for the last 1 h of a 2-h MG132 treatment and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with BTB-1 plotted as the distance from the spindle equator. See also Video 4. (C) DAP measurements of kinetochore position in HeLa cells treated as in B. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (D) Hec1-S55 phosphorylation in HeLa cells during metaphase in the presence of BTB-1. Cells were treated with BTB-1 and either DMSO or AZD1152 and/or MLN8237 for the last 1 h of a 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). (E) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells treated as in D, which was calculated and displayed as box and dot plots as in Fig. 2 B. The median of cells treated with BTB-1 without Aurora inhibitors was set as 1. The data represent a minimum of 347 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. (F) Trajectories of kinetochores in HeLa cells arrested in metaphase in the presence or absence of Calyculin A. Cells were treated with or without Calyculin A for the last 1 h of a 2-h MG132 treatment, then observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with Calyculin A, plotted as the distance from the spindle equator. See also Video 5. (G) DAP measurements of kinetochore position in RPE-1 cells treated as in F. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (H) Hec1-S69 phosphorylation in HeLa cells during metaphase in the presence of BTB-1 or UMK57, a MCAK potentiator. Cells were treated with BTB-1 or UMK57 for the last 1 h of a 3-h MG132 treatment, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S69 (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). (I) Quantification of the Hec1-S69 phosphorylation signal in HeLa cells treated as in H, which was calculated and displayed as in Fig. 2 B. The median of DMSO-treated cells was set as 1. The data represent a minimum of 259 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (J) Expression of Hec1 constructs in RPE-1 cells depleted of endogenous Hec1. Lysate of Hec1-depleted cells transfected with each Hec1 construct was subjected to immunoblot analysis using antibodies as indicated. (K) Kinetochore–microtubule attachments in metaphase HeLa cells. HeLa cells expressing EGFP–CENP-A were subjected to immunostaining with an antibody against GFP (green) and α-tubulin (red). DNA was stained with DAPI (blue). Magnified view of kinetochore–microtubule attachments in selected Z-slices are shown in insets, where merotelic attachment was judged by the presence of microtubules between sister kinetochores. Scale bar: 5 µm (white), 500 nm (yellow). (L) Quantification of the kinetochore pairs forming merotelic attachment in HeLa cells arrested in metaphase in the presence of BTB-1 or UMK57. HeLa cells expressing EGFP–CENP-A were fixed and stained as in K. The data represent the percentage of merotelic attachment, and error bars represent SD of three independent experiments in which a minimum of 100 kinetochore–microtubule attachments in a minimum of 10 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (M) Quantification of the kinetochore pairs in RPE-1 cells arrested in metaphase forming merotelic attachment in the presence of AZD1152 or MLN8237. RPE-1 cells expressing EGFP–CENP-A were fixed and stained as in K. The data represent the percentage of merotelic attachment, and error bars represent SD of three independent experiments in which a minimum of 100 kinetochore–microtubule attachments in a minimum of 10 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (N) Spindle morphology of RPE-1 cells treated with or without Aurora inhibitors. Cells were treated with either DMSO or AZD1152 and/or MLN8237 for the last 1 h of the 3-h MG132 treatment, then incubated on ice for 0 or 10 min before fixation and stained with an anti–α-tubulin antibody (α-tub; green). DNA was stained with DAPI (blue). Scale bars: 5 µm. (O) Quantification of microtubule signals on the spindle. The α-tubulin signal intensities on the spindle of cells treated as in N were measured. The intensity of DMSO-treated cells without cold treatment 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. (P) Expression of Aurora A and TPX2 on the spindle in nontransformed and cancer cell lines during metaphase. Cells were treated with MG132 for 3 h and then fixed and stained with anti–Aurora A (AurA; red) and anti-TPX2 (green) antibodies. DNA was stained with DAPI (blue). Scale bar: 5 µm. Signal intensity of Aurora A and TPX2 and the signal ratio of Aurora A to TPX2 are shown in the right graphs. The average values in RPE-1 cells were set as 1. Error bars represent SD of five independent experiments in which a minimum of 10 cells were observed for each cell line. P value was obtained using the Tukey-Kramer multiple comparisons test.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal