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    5-ITu treatment prevents accumulation and activity of Aurora B at centromeres, but not kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM 5-ITu or 10 μM 5-ITu plus 10 μM <t>BAY320</t> for 30 min. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of fluorescence intensities. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; four experiments); 5-ITu ( n = 161 centromeres; 14 cells; three experiments); 5-ITu + BAY320 ( n = 115 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); 5-ITu ( n = 210 centromeres; 10 cells; three experiments); 5-ITu + BAY320 ( n = 195 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); 5-ITu ( n = 359 kinetochores; 38 cells; four experiments); 5-ITu + BAY320 ( n = 279 kinetochores; 40 cells; four experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); 5-ITu ( n = 374 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 336 kinetochores; 16 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); 5-ITu ( n = 337 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 309 kinetochores; 18 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); 5-ITu ( n = 295 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 315 kinetochores; 16 cells; three experiments). Error bars represent SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test condition compared with control cells. Scale bars, 10 µm. AU, arbitrary units.
    Bay320, supplied by Bayer AG, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells"

    Article Title: Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201905144

    5-ITu treatment prevents accumulation and activity of Aurora B at centromeres, but not kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM 5-ITu or 10 μM 5-ITu plus 10 μM BAY320 for 30 min. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of fluorescence intensities. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; four experiments); 5-ITu ( n = 161 centromeres; 14 cells; three experiments); 5-ITu + BAY320 ( n = 115 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); 5-ITu ( n = 210 centromeres; 10 cells; three experiments); 5-ITu + BAY320 ( n = 195 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); 5-ITu ( n = 359 kinetochores; 38 cells; four experiments); 5-ITu + BAY320 ( n = 279 kinetochores; 40 cells; four experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); 5-ITu ( n = 374 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 336 kinetochores; 16 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); 5-ITu ( n = 337 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 309 kinetochores; 18 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); 5-ITu ( n = 295 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 315 kinetochores; 16 cells; three experiments). Error bars represent SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test condition compared with control cells. Scale bars, 10 µm. AU, arbitrary units.
    Figure Legend Snippet: 5-ITu treatment prevents accumulation and activity of Aurora B at centromeres, but not kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM 5-ITu or 10 μM 5-ITu plus 10 μM BAY320 for 30 min. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of fluorescence intensities. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; four experiments); 5-ITu ( n = 161 centromeres; 14 cells; three experiments); 5-ITu + BAY320 ( n = 115 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); 5-ITu ( n = 210 centromeres; 10 cells; three experiments); 5-ITu + BAY320 ( n = 195 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); 5-ITu ( n = 359 kinetochores; 38 cells; four experiments); 5-ITu + BAY320 ( n = 279 kinetochores; 40 cells; four experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); 5-ITu ( n = 374 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 336 kinetochores; 16 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); 5-ITu ( n = 337 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 309 kinetochores; 18 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); 5-ITu ( n = 295 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 315 kinetochores; 16 cells; three experiments). Error bars represent SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test condition compared with control cells. Scale bars, 10 µm. AU, arbitrary units.

    Techniques Used: Activity Assay, Immunofluorescence, Control, Staining, Fluorescence

    Specificity of Bub1 and Haspin kinase inhibitors. (A) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH3-T3. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 16 cells from three independent experiments were measured. Error bars indicate SD. (B) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH2A-T120. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 14 cells from three independent experiments were measured. Error bars indicate SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between each experiment and control cells. Scale bars, 10 µm. AU, arbitrary units.
    Figure Legend Snippet: Specificity of Bub1 and Haspin kinase inhibitors. (A) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH3-T3. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 16 cells from three independent experiments were measured. Error bars indicate SD. (B) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH2A-T120. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 14 cells from three independent experiments were measured. Error bars indicate SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between each experiment and control cells. Scale bars, 10 µm. AU, arbitrary units.

    Techniques Used: Immunofluorescence, Control, Fluorescence

    Bub1 kinase activity is not required for localization or activity of Aurora B at kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM Bub1 kinase inhibitor BAY320 or 20 μM Aurora B kinase inhibitor ZM447439. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of centromere or kinetochore fluorescence intensity. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; three experiments); BAY320 ( n = 226 centromeres; 13 cells; four experiments); ZM447439 ( n = 222 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); BAY320 ( n = 245 centromeres; 10 cells; three experiments); ZM447439 ( n = 179 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); BAY320 ( n = 350 kinetochores; 24 cells; four experiments); ZM447439 ( n = 219 kinetochores; 6 cells; three experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); BAY320 ( n = 272 kinetochores; 15 cells; three experiments); ZM447439 ( n = 224 kinetochores; 11 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); BAY320 ( n = 348 kinetochores; 17 cells; three experiments); ZM447439 ( n = 281 kinetochores; 14 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); BAY320 ( n = 368 kinetochores; 17 cells; three experiments); ZM447439 ( n = 209 kinetochores; 10 cells; two experiments). (G) Quantification of time (in min) to anaphase onset after washout of monastrol. HeLa cells were treated with 150 μM monastrol for 2 h. Cells were washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. The time from initiation of washout to anaphase onset was scored. For each condition, at least 200 cells were quantified from three experiments. (H) Quantification of chromosome segregation errors in cells treated with 150 μM monastrol for 2 h, and washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. Errors in chromosome segregation were quantified from anaphase cells. For A-F, significance values were calculated from unpaired nonparametric Student’s t tests. For G and H, significance values were calculated from one-way ordinary ANOVA tests. AU, arbitrary units; Cont, control; BAY, BAY320; ZM, ZM447439. Scale bars, 10 µm.
    Figure Legend Snippet: Bub1 kinase activity is not required for localization or activity of Aurora B at kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM Bub1 kinase inhibitor BAY320 or 20 μM Aurora B kinase inhibitor ZM447439. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of centromere or kinetochore fluorescence intensity. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; three experiments); BAY320 ( n = 226 centromeres; 13 cells; four experiments); ZM447439 ( n = 222 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); BAY320 ( n = 245 centromeres; 10 cells; three experiments); ZM447439 ( n = 179 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); BAY320 ( n = 350 kinetochores; 24 cells; four experiments); ZM447439 ( n = 219 kinetochores; 6 cells; three experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); BAY320 ( n = 272 kinetochores; 15 cells; three experiments); ZM447439 ( n = 224 kinetochores; 11 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); BAY320 ( n = 348 kinetochores; 17 cells; three experiments); ZM447439 ( n = 281 kinetochores; 14 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); BAY320 ( n = 368 kinetochores; 17 cells; three experiments); ZM447439 ( n = 209 kinetochores; 10 cells; two experiments). (G) Quantification of time (in min) to anaphase onset after washout of monastrol. HeLa cells were treated with 150 μM monastrol for 2 h. Cells were washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. The time from initiation of washout to anaphase onset was scored. For each condition, at least 200 cells were quantified from three experiments. (H) Quantification of chromosome segregation errors in cells treated with 150 μM monastrol for 2 h, and washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. Errors in chromosome segregation were quantified from anaphase cells. For A-F, significance values were calculated from unpaired nonparametric Student’s t tests. For G and H, significance values were calculated from one-way ordinary ANOVA tests. AU, arbitrary units; Cont, control; BAY, BAY320; ZM, ZM447439. Scale bars, 10 µm.

    Techniques Used: Activity Assay, Immunofluorescence, Control, Staining, Fluorescence



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    bay320  (Bayer AG)
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    Bayer AG bay320
    5-ITu treatment prevents accumulation and activity of Aurora B at centromeres, but not kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM 5-ITu or 10 μM 5-ITu plus 10 μM <t>BAY320</t> for 30 min. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of fluorescence intensities. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; four experiments); 5-ITu ( n = 161 centromeres; 14 cells; three experiments); 5-ITu + BAY320 ( n = 115 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); 5-ITu ( n = 210 centromeres; 10 cells; three experiments); 5-ITu + BAY320 ( n = 195 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); 5-ITu ( n = 359 kinetochores; 38 cells; four experiments); 5-ITu + BAY320 ( n = 279 kinetochores; 40 cells; four experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); 5-ITu ( n = 374 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 336 kinetochores; 16 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); 5-ITu ( n = 337 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 309 kinetochores; 18 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); 5-ITu ( n = 295 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 315 kinetochores; 16 cells; three experiments). Error bars represent SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test condition compared with control cells. Scale bars, 10 µm. AU, arbitrary units.
    Bay320, supplied by Bayer AG, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bay320/product/Bayer AG
    Average 90 stars, based on 1 article reviews
    bay320 - by Bioz Stars, 2026-02
    90/100 stars
    		  Buy from Supplier

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    5-ITu treatment prevents accumulation and activity of Aurora B at centromeres, but not kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM 5-ITu or 10 μM 5-ITu plus 10 μM BAY320 for 30 min. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of fluorescence intensities. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; four experiments); 5-ITu ( n = 161 centromeres; 14 cells; three experiments); 5-ITu + BAY320 ( n = 115 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); 5-ITu ( n = 210 centromeres; 10 cells; three experiments); 5-ITu + BAY320 ( n = 195 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); 5-ITu ( n = 359 kinetochores; 38 cells; four experiments); 5-ITu + BAY320 ( n = 279 kinetochores; 40 cells; four experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); 5-ITu ( n = 374 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 336 kinetochores; 16 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); 5-ITu ( n = 337 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 309 kinetochores; 18 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); 5-ITu ( n = 295 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 315 kinetochores; 16 cells; three experiments). Error bars represent SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test condition compared with control cells. Scale bars, 10 µm. AU, arbitrary units.

    Journal: The Journal of Cell Biology

    Article Title: Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells

    doi: 10.1083/jcb.201905144

    Figure Lengend Snippet: 5-ITu treatment prevents accumulation and activity of Aurora B at centromeres, but not kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM 5-ITu or 10 μM 5-ITu plus 10 μM BAY320 for 30 min. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of fluorescence intensities. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; four experiments); 5-ITu ( n = 161 centromeres; 14 cells; three experiments); 5-ITu + BAY320 ( n = 115 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); 5-ITu ( n = 210 centromeres; 10 cells; three experiments); 5-ITu + BAY320 ( n = 195 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); 5-ITu ( n = 359 kinetochores; 38 cells; four experiments); 5-ITu + BAY320 ( n = 279 kinetochores; 40 cells; four experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); 5-ITu ( n = 374 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 336 kinetochores; 16 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); 5-ITu ( n = 337 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 309 kinetochores; 18 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); 5-ITu ( n = 295 kinetochores; 16 cells; three experiments); 5-ITu + BAY320 ( n = 315 kinetochores; 16 cells; three experiments). Error bars represent SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test condition compared with control cells. Scale bars, 10 µm. AU, arbitrary units.

    Article Snippet: For inhibition of Bub1 kinase, cells were treated with 10 μM BAY320 (provided by G. Siemeister, Bayer AG, Berlin, Germany) 30 min before fixation.

    Techniques: Activity Assay, Immunofluorescence, Control, Staining, Fluorescence

    Specificity of Bub1 and Haspin kinase inhibitors. (A) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH3-T3. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 16 cells from three independent experiments were measured. Error bars indicate SD. (B) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH2A-T120. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 14 cells from three independent experiments were measured. Error bars indicate SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between each experiment and control cells. Scale bars, 10 µm. AU, arbitrary units.

    Journal: The Journal of Cell Biology

    Article Title: Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells

    doi: 10.1083/jcb.201905144

    Figure Lengend Snippet: Specificity of Bub1 and Haspin kinase inhibitors. (A) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH3-T3. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 16 cells from three independent experiments were measured. Error bars indicate SD. (B) Immunofluorescence images of control HeLa cells and cells treated with 10 μM BAY320, 10 μM 5-ITu, or both kinase inhibitors for 30 min and then immunostained for pH2A-T120. Quantification of whole cell fluorescence intensity for each condition is shown to the right. Fluorescence intensities for test conditions were normalized to those calculated from control cells. For all conditions, at least 14 cells from three independent experiments were measured. Error bars indicate SD. Significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between each experiment and control cells. Scale bars, 10 µm. AU, arbitrary units.

    Article Snippet: For inhibition of Bub1 kinase, cells were treated with 10 μM BAY320 (provided by G. Siemeister, Bayer AG, Berlin, Germany) 30 min before fixation.

    Techniques: Immunofluorescence, Control, Fluorescence

    Bub1 kinase activity is not required for localization or activity of Aurora B at kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM Bub1 kinase inhibitor BAY320 or 20 μM Aurora B kinase inhibitor ZM447439. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of centromere or kinetochore fluorescence intensity. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; three experiments); BAY320 ( n = 226 centromeres; 13 cells; four experiments); ZM447439 ( n = 222 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); BAY320 ( n = 245 centromeres; 10 cells; three experiments); ZM447439 ( n = 179 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); BAY320 ( n = 350 kinetochores; 24 cells; four experiments); ZM447439 ( n = 219 kinetochores; 6 cells; three experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); BAY320 ( n = 272 kinetochores; 15 cells; three experiments); ZM447439 ( n = 224 kinetochores; 11 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); BAY320 ( n = 348 kinetochores; 17 cells; three experiments); ZM447439 ( n = 281 kinetochores; 14 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); BAY320 ( n = 368 kinetochores; 17 cells; three experiments); ZM447439 ( n = 209 kinetochores; 10 cells; two experiments). (G) Quantification of time (in min) to anaphase onset after washout of monastrol. HeLa cells were treated with 150 μM monastrol for 2 h. Cells were washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. The time from initiation of washout to anaphase onset was scored. For each condition, at least 200 cells were quantified from three experiments. (H) Quantification of chromosome segregation errors in cells treated with 150 μM monastrol for 2 h, and washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. Errors in chromosome segregation were quantified from anaphase cells. For A-F, significance values were calculated from unpaired nonparametric Student’s t tests. For G and H, significance values were calculated from one-way ordinary ANOVA tests. AU, arbitrary units; Cont, control; BAY, BAY320; ZM, ZM447439. Scale bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells

    doi: 10.1083/jcb.201905144

    Figure Lengend Snippet: Bub1 kinase activity is not required for localization or activity of Aurora B at kinetochores. (A – F) Immunofluorescence images of control HeLa cells or HeLa cells treated with either 10 μM Bub1 kinase inhibitor BAY320 or 20 μM Aurora B kinase inhibitor ZM447439. Cells were fixed and stained with the indicated antibodies. To the right of each immunofluorescence panel is the quantification of centromere or kinetochore fluorescence intensity. Fluorescence intensities for test conditions were normalized to those calculated from control cells. n values for each experiment follow. ABK: Control ( n = 153 centromeres; 15 cells; three experiments); BAY320 ( n = 226 centromeres; 13 cells; four experiments); ZM447439 ( n = 222 centromeres; 11 cells; three experiments). pINCENP: Control ( n = 349 centromeres; 15 cells; three experiments); BAY320 ( n = 245 centromeres; 10 cells; three experiments); ZM447439 ( n = 179 centromeres; 10 cells; three experiments). pABK-T232: Control ( n = 621 kinetochores; 42 cells; four experiments); BAY320 ( n = 350 kinetochores; 24 cells; four experiments); ZM447439 ( n = 219 kinetochores; 6 cells; three experiments). pHec1-S44: Control ( n = 384 kinetochores; 16 cells; three experiments); BAY320 ( n = 272 kinetochores; 15 cells; three experiments); ZM447439 ( n = 224 kinetochores; 11 cells; three experiments). pKnl1-S24: Control ( n = 363 kinetochores; 16 cells; three experiments); BAY320 ( n = 348 kinetochores; 17 cells; three experiments); ZM447439 ( n = 281 kinetochores; 14 cells; three experiments). pDsn1-S109: Control ( n = 400 kinetochores; 18 cells; three experiments); BAY320 ( n = 368 kinetochores; 17 cells; three experiments); ZM447439 ( n = 209 kinetochores; 10 cells; two experiments). (G) Quantification of time (in min) to anaphase onset after washout of monastrol. HeLa cells were treated with 150 μM monastrol for 2 h. Cells were washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. The time from initiation of washout to anaphase onset was scored. For each condition, at least 200 cells were quantified from three experiments. (H) Quantification of chromosome segregation errors in cells treated with 150 μM monastrol for 2 h, and washed out into control media, or media containing either 10 μM BAY320 or 20 μM ZM447439. Errors in chromosome segregation were quantified from anaphase cells. For A-F, significance values were calculated from unpaired nonparametric Student’s t tests. For G and H, significance values were calculated from one-way ordinary ANOVA tests. AU, arbitrary units; Cont, control; BAY, BAY320; ZM, ZM447439. Scale bars, 10 µm.

    Article Snippet: For inhibition of Bub1 kinase, cells were treated with 10 μM BAY320 (provided by G. Siemeister, Bayer AG, Berlin, Germany) 30 min before fixation.

    Techniques: Activity Assay, Immunofluorescence, Control, Staining, Fluorescence