Figure S3.
Hec1-S55 phosphorylation in RPE-1 cells during metaphase is not dependent on Aurora B.(A) Schematic of the procedure of experiments using cells expressing Aurora A/B–mAID-mClover or mAID-mClover-TPX2. (B) Depletion of Aurora A/B–mAID-mClover by adding IAA in RPE-1 cells arrested in metaphase. RPE-1 cells containing Aurora A/B–mAID-mClover were treated as in A, then harvested and lysed for immunoblot analysis using antibodies as indicated. Parental RPE-1 cells were used as a control. (C) Schematic diagram of tagging endogenous Aurora B with mAID-mClover tag by the CRISPR/Cas–based method. (D) Depletion of Aurora B–mAID-mClover by adding IAA to RPE-1 cells arrested in metaphase. Cells were treated as in A, then fixed and stained with an anti–Aurora B (AurB; red) antibody. DNA was stained with DAPI (blue). The mClover signal (green) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Scale bar: 5 µm. (E) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase depleted of Aurora B using the AID system. Cells treated as in A with or without AZD1152 and/or MLN8237 were fixed and stained with anti-Hec1 (blue, shown as green), and anti–phospho-Hec1-S55 (red) antibodies. The mClover signal (green, shown as monochrome) was acquired by fluorescence microscopy. 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 arrested in metaphase depleted of Aurora B using the AID system. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in E was calculated and displayed as box and dot plots as in Fig. 2 B. The median of DMSO-treated cells was set as 1. The data represent a minimum of 245 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (G) Efficiency of INCENP RNAi. Lysate of cells transfected with an siRNA against INCENP was subjected to immunoblot analysis using antibodies as indicated. (H) Hec1-S55 and -S69 phosphorylation in RPE-1 cells depleted of INCENP in metaphase. Cells were fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (upper panels, red) or -S69 (lower panels, 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). (I) Quantification of the Hec1-S55 and -S69 phosphorylation signals in RPE-1 cells depleted of INCENP in metaphase. Relative intensity of the phosphorylated Hec1-S55 (left graph) or -S69 (right graph) 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 cells treated with control siRNA was set as 1. The data represent a minimum of 322 kinetochores from five cells for each condition. P values were obtained using the Mann–Whitney U test. AURKA, Aurora kinase A; AURKB, Aurora kinase B; Dox, doxycycline; Controlsi, treatment with a control siRNA; INCENPsi, treatment with an siRNA for INCENP.

Hec1-S55 phosphorylation in RPE-1 cells during metaphase is not dependent on Aurora B. (A) Schematic of the procedure of experiments using cells expressing Aurora A/B–mAID-mClover or mAID-mClover-TPX2. (B) Depletion of Aurora A/B–mAID-mClover by adding IAA in RPE-1 cells arrested in metaphase. RPE-1 cells containing Aurora A/B–mAID-mClover were treated as in A, then harvested and lysed for immunoblot analysis using antibodies as indicated. Parental RPE-1 cells were used as a control. (C) Schematic diagram of tagging endogenous Aurora B with mAID-mClover tag by the CRISPR/Cas–based method. (D) Depletion of Aurora B–mAID-mClover by adding IAA to RPE-1 cells arrested in metaphase. Cells were treated as in A, then fixed and stained with an anti–Aurora B (AurB; red) antibody. DNA was stained with DAPI (blue). The mClover signal (green) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Scale bar: 5 µm. (E) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase depleted of Aurora B using the AID system. Cells treated as in A with or without AZD1152 and/or MLN8237 were fixed and stained with anti-Hec1 (blue, shown as green), and anti–phospho-Hec1-S55 (red) antibodies. The mClover signal (green, shown as monochrome) was acquired by fluorescence microscopy. 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 arrested in metaphase depleted of Aurora B using the AID system. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in E was calculated and displayed as box and dot plots as in Fig. 2 B. The median of DMSO-treated cells was set as 1. The data represent a minimum of 245 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (G) Efficiency of INCENP RNAi. Lysate of cells transfected with an siRNA against INCENP was subjected to immunoblot analysis using antibodies as indicated. (H) Hec1-S55 and -S69 phosphorylation in RPE-1 cells depleted of INCENP in metaphase. Cells were fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (upper panels, red) or -S69 (lower panels, 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). (I) Quantification of the Hec1-S55 and -S69 phosphorylation signals in RPE-1 cells depleted of INCENP in metaphase. Relative intensity of the phosphorylated Hec1-S55 (left graph) or -S69 (right graph) 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 cells treated with control siRNA was set as 1. The data represent a minimum of 322 kinetochores from five cells for each condition. P values were obtained using the Mann–Whitney U test. AURKA, Aurora kinase A; AURKB, Aurora kinase B; Dox, doxycycline; Controlsi, treatment with a control siRNA; INCENPsi, treatment with an siRNA for INCENP.

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