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aurora b protein (Boster Bio)

Name: aurora b protein
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Boster Bio aurora b protein
Aurora B Protein, supplied by Boster Bio, 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/aurora b protein/product/Boster Bio
Average 90 stars, based on 1 article reviews

aurora b protein - by Bioz Stars, 2026-02

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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: pH3-T3 and pH2A-T120 are each capable of recruiting Aurora B kinase and the CPC. (A and B) Immunofluorescence images of U2OS cells expressing LacI-GFP-Bub1 (A) or LacI-BFP-Haspin (B). Cells were immunostained for the indicated centromere/kinetochore proteins or phospho-specific epitopes. Arrows point to the LacO array with indicated LacI-fused protein shown in the insets. Cells were synchronized in G2 using RO-3306 and washed out of drug 30 min before fixation to enrich for early mitotic cells. (C and D) Quantification of experiments in A and B. The average fluorescence intensity of each antibody was measured at the LacO array in cells expressing either LacI-BFP or LacI-GFP (not fused to a test protein), and average values were measured and used to calculate thresholds. Fluorescence intensities of each antibody were then measured in cells expressing LacI-BFP-Haspin or LacI-GFP-Bub1, and cells were scored as positive if intensity values were higher than the calculated thresholds. For each experiment, ≥20 cells were quantified from n ≥ 3 experiments. Error bars represent SD. Significance values were calculated from a one-way ordinary ANOVA test. Shown are significance values between experiments for each test antibody and either pH2A-T120 (C) or pH3-T3 (D). Scale bars, 10 µm (whole cells) and 1 µm (insets). n.s., not significant.

Article Snippet: For the phosphatase assay, 1.7 μM Aurora B/INCENP complex was incubated with or without lambda protein phosphatase (2,400 U; NEB P0753S) in 1× NEBuffer for protein metallophosphatases and 1 mM MnCl 2 , for 30 min at 30°C.

Techniques: Immunofluorescence, Expressing, Fluorescence

Sgo1 recruits the CPC to an ectopic chromosome locus in human cells. (A) Immunofluorescence images of U2OS cells expressing LacI-GFP-Sgo1. Cells were immunostained for the indicated proteins or phospho-histone epitopes and analyzed for positive recruitment based on a threshold intensity determined from U2OS cells expressing LacI-GFP. (B) Quantification of the recruitment of indicated proteins or phospho-epitopes in LacI-GFP-Sgo1–expressing cells. (C) Quantification of pH2A-T120 recruitment in Bub1 or Sgo1-LacI–expressing cells. For all conditions, cells were synchronized in G2 using RO-3306 and washed out of drug 30 min before fixation to enrich for mitotic cells. (D) Immunofluorescence images of U2OS cells expressing LacI-GFP-Bub1. Top row: control; bottom row: Sgo1 siRNA. (E) Quantification of percent Sgo1-positive cells (left) and percent Aurora B–positive cells (right) in control and Sgo1 siRNA-treated cells. For each condition shown, ≥20 cells were quantified from at least two independent experiments. Error bars represent SD. For B, significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test antibody and pH2A-T120. For C, the significance value was calculated using an unpaired nonparametric Student’s t test. 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: Sgo1 recruits the CPC to an ectopic chromosome locus in human cells. (A) Immunofluorescence images of U2OS cells expressing LacI-GFP-Sgo1. Cells were immunostained for the indicated proteins or phospho-histone epitopes and analyzed for positive recruitment based on a threshold intensity determined from U2OS cells expressing LacI-GFP. (B) Quantification of the recruitment of indicated proteins or phospho-epitopes in LacI-GFP-Sgo1–expressing cells. (C) Quantification of pH2A-T120 recruitment in Bub1 or Sgo1-LacI–expressing cells. For all conditions, cells were synchronized in G2 using RO-3306 and washed out of drug 30 min before fixation to enrich for mitotic cells. (D) Immunofluorescence images of U2OS cells expressing LacI-GFP-Bub1. Top row: control; bottom row: Sgo1 siRNA. (E) Quantification of percent Sgo1-positive cells (left) and percent Aurora B–positive cells (right) in control and Sgo1 siRNA-treated cells. For each condition shown, ≥20 cells were quantified from at least two independent experiments. Error bars represent SD. For B, significance values were calculated using a one-way ordinary ANOVA test. Shown are significance values between experiments for each test antibody and pH2A-T120. For C, the significance value was calculated using an unpaired nonparametric Student’s t test. Scale bars, 10 µm.

Techniques: Immunofluorescence, Expressing

The Bub1/pH2A-T120/Sgo1 pathway contributes to the accumulation of centromere-associated Aurora B but not localization or activity of kinetochore-associated Aurora B. (A, C, E, and G) Immunofluorescence images of HeLa cells depleted of Sgo1 and stained for either Aurora B (ABK), active phosphorylated ABK (pABK-T232), phosphorylated Hec1-S44 (pHec1-S44), or Sgo1. (B, D, F, and H) Quantification of centromere or kinetochore fluorescence intensities of Aurora B, pABK-T232, pHec1-S44, and Sgo1. For all conditions, at least 120 kinetochores from 10 cells were measured from a total of three independent experiments. Fluorescence intensities for Sgo1 depletion were normalized to those calculated from control cells. (I, K, and M) HeLa Flp-In TREx cells were induced to express GFP-Sgo1-WT, GFP-Sgo1-K492A, or GFP-Sgo1-T346A fusion proteins and immunostained for ABK, pABK-T232, or pHec1-S44. (J, L, and N) Quantification of centromere (ABK) or kinetochore (pABK-T232, pHec1-S44) normalized fluorescence intensities. For ABK quantification, 120 centromeres were measured from 15 cells from three independent experiments. For pABK-T232 and pHec1-S44, at least 200 kinetochores were measured from at least 15 cells from three independent experiments. Fluorescence intensities for test conditions were normalized to those calculated from control cells. Error bars represent SD. For B, D, F, and H, significance values were calculated from unpaired nonparametric Student's t tests. For J, L, and N, significance values were calculated from a one-way ordinary ANOVA test, and shown are significance values between experiments using cells overexpressing WT or mutant Sgo1 constructs compared with control, untreated cells. AU, arbitrary units; ACA, anti-centromere antibody. 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: The Bub1/pH2A-T120/Sgo1 pathway contributes to the accumulation of centromere-associated Aurora B but not localization or activity of kinetochore-associated Aurora B. (A, C, E, and G) Immunofluorescence images of HeLa cells depleted of Sgo1 and stained for either Aurora B (ABK), active phosphorylated ABK (pABK-T232), phosphorylated Hec1-S44 (pHec1-S44), or Sgo1. (B, D, F, and H) Quantification of centromere or kinetochore fluorescence intensities of Aurora B, pABK-T232, pHec1-S44, and Sgo1. For all conditions, at least 120 kinetochores from 10 cells were measured from a total of three independent experiments. Fluorescence intensities for Sgo1 depletion were normalized to those calculated from control cells. (I, K, and M) HeLa Flp-In TREx cells were induced to express GFP-Sgo1-WT, GFP-Sgo1-K492A, or GFP-Sgo1-T346A fusion proteins and immunostained for ABK, pABK-T232, or pHec1-S44. (J, L, and N) Quantification of centromere (ABK) or kinetochore (pABK-T232, pHec1-S44) normalized fluorescence intensities. For ABK quantification, 120 centromeres were measured from 15 cells from three independent experiments. For pABK-T232 and pHec1-S44, at least 200 kinetochores were measured from at least 15 cells from three independent experiments. Fluorescence intensities for test conditions were normalized to those calculated from control cells. Error bars represent SD. For B, D, F, and H, significance values were calculated from unpaired nonparametric Student's t tests. For J, L, and N, significance values were calculated from a one-way ordinary ANOVA test, and shown are significance values between experiments using cells overexpressing WT or mutant Sgo1 constructs compared with control, untreated cells. AU, arbitrary units; ACA, anti-centromere antibody. Scale bars, 10 µm.

Techniques: Activity Assay, Immunofluorescence, Staining, Fluorescence, Mutagenesis, Construct

Depletion of Bub1 protein results in reduction of both centromere and kinetochore-associated Aurora B in human cells. (A, D, G, and I) Immunofluorescence images of HeLa cells depleted of Bub1 and immunostained for the indicated antibodies. (B, C, E, F, H, and J) Quantification of kinetochore or centromere fluorescence intensities of indicated antibodies. Error bars indicate SD. For all conditions, at least 120 kinetochores from 10 cells were measured from a total of three independent experiments. Error bars represent SD. Significance values were calculated using unpaired nonparametric Student's t tests. 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: Depletion of Bub1 protein results in reduction of both centromere and kinetochore-associated Aurora B in human cells. (A, D, G, and I) Immunofluorescence images of HeLa cells depleted of Bub1 and immunostained for the indicated antibodies. (B, C, E, F, H, and J) Quantification of kinetochore or centromere fluorescence intensities of indicated antibodies. Error bars indicate SD. For all conditions, at least 120 kinetochores from 10 cells were measured from a total of three independent experiments. Error bars represent SD. Significance values were calculated using unpaired nonparametric Student's t tests. Scale bars, 10 µm. AU, arbitrary units.

Techniques: Immunofluorescence, Fluorescence

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.

Techniques: Activity Assay, Immunofluorescence, Staining, 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.

Techniques: Activity Assay, Immunofluorescence, Staining, Fluorescence

Characterization of pINCENP-S893,894 antibody. (A) Immunofluorescence images of HeLa cells in various stages of mitosis stained with pINCENP-S893, S894, Aurora B, and ACA. Arrows point to the kinetochore pairs shown in the insets on the right. (B) Immunofluorescence images of a control HeLa cell and a HeLa cell treated with 10 μM ZM447439. (C) Immunofluorescence images of a control cell and HeLa cell treated with INCENP siRNA. (D) A complex of Aurora B (amino acids 60–361) and INCENP (amino acids 1–68 and 834–900) was incubated with or without lambda protein phosphatase (pptase), and reactions were terminated with the addition of SDS-PAGE sample buffer. Images show Western blots of reactions probed with pINCENP-S893, S894 (left) and pABK-T232 (middle) antibodies (ab). A Coomassie-stained gel is shown to the right. Molecular weight (MW) markers are indicated. Scale bars, 10 µm (whole cells) and 1 µm (insets). Whole cell images shown are maximum intensity projections of z-stacks containing 64 planes, and inset images shown are projections from 2 to 4 planes.

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: Characterization of pINCENP-S893,894 antibody. (A) Immunofluorescence images of HeLa cells in various stages of mitosis stained with pINCENP-S893, S894, Aurora B, and ACA. Arrows point to the kinetochore pairs shown in the insets on the right. (B) Immunofluorescence images of a control HeLa cell and a HeLa cell treated with 10 μM ZM447439. (C) Immunofluorescence images of a control cell and HeLa cell treated with INCENP siRNA. (D) A complex of Aurora B (amino acids 60–361) and INCENP (amino acids 1–68 and 834–900) was incubated with or without lambda protein phosphatase (pptase), and reactions were terminated with the addition of SDS-PAGE sample buffer. Images show Western blots of reactions probed with pINCENP-S893, S894 (left) and pABK-T232 (middle) antibodies (ab). A Coomassie-stained gel is shown to the right. Molecular weight (MW) markers are indicated. Scale bars, 10 µm (whole cells) and 1 µm (insets). Whole cell images shown are maximum intensity projections of z-stacks containing 64 planes, and inset images shown are projections from 2 to 4 planes.

Techniques: Immunofluorescence, Staining, Incubation, SDS Page, Western Blot, Molecular Weight

Inhibition of Haspin kinase results in relocalization of Aurora B from inner centromeres to kinetochore-proximal outer centromeres. (A) Immunofluorescence images of control and 5 μM 5-ITu-treated early prometaphase HeLa cells expressing Aurora B kinase-GFP (ABK-GFP) and stained with antibodies to CENP-C. Arrows point to the kinetochore pairs shown in the insets. Linescans through the centromere/kinetochore region are shown to the right of each panel. (B) Immunofluorescence images of control and 5 μM 5-ITu-treated early prometaphase HeLa cells expressing ABK-GFP and stained with antibodies to pABK-T232. Arrows point to the kinetochore pairs shown in the insets. Linescans through the centromere/kinetochore region are shown to the right of each panel. Scale bars, 10 µm (whole cells) and 1 µm (insets). Whole cell images shown are maximum intensity projections of z-stacks containing 64 planes, and inset images shown are projections from 2 to 4 planes. 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: Inhibition of Haspin kinase results in relocalization of Aurora B from inner centromeres to kinetochore-proximal outer centromeres. (A) Immunofluorescence images of control and 5 μM 5-ITu-treated early prometaphase HeLa cells expressing Aurora B kinase-GFP (ABK-GFP) and stained with antibodies to CENP-C. Arrows point to the kinetochore pairs shown in the insets. Linescans through the centromere/kinetochore region are shown to the right of each panel. (B) Immunofluorescence images of control and 5 μM 5-ITu-treated early prometaphase HeLa cells expressing ABK-GFP and stained with antibodies to pABK-T232. Arrows point to the kinetochore pairs shown in the insets. Linescans through the centromere/kinetochore region are shown to the right of each panel. Scale bars, 10 µm (whole cells) and 1 µm (insets). Whole cell images shown are maximum intensity projections of z-stacks containing 64 planes, and inset images shown are projections from 2 to 4 planes. AU, arbitrary units.

Techniques: Inhibition, Immunofluorescence, Expressing, Staining

Aurora B kinase and INCENP localize to both centromeres and kinetochores in mitotic cells. (A) Immunofluorescence images of an early prometaphase control HeLa cell stained with antibodies to pABK-T232, CENP-C, and the N-terminus of Hec1 (antibody 9G3). Arrows point to the kinetochore pairs shown in the insets. A linescan through the centromere/kinetochore region is shown to the right of the panel. (B) Immunofluorescence images of an early prometaphase control HeLa cell stained with antibodies to pINCENP, CENP-C, and the N terminus of Hec1 (antibody 9G3). Numbers indicate kinetochore pairs shown in the insets, and linescans through the centromere/kinetochore region are shown to the right of each panel. pINCENP is localized to the kinetochore (pair 1), the centromere and kinetochore (pair 2), or the kinetochore-proximal outer centromere (pair 3). (C) Plots of the mean distance between the indicated proteins. Each point on the graph represents a distance measurement for a pair of sister chromatids, and the mean value is indicated. Error bars indicate SD. n values are listed in . (D) Summary of distance measurements from experiments in the study. Scale bars, 10 µm (whole cells) and 1 µm (insets). Whole cell images shown are maximum intensity projections of z-stacks containing 64 planes, and inset images shown are projections from 2 to 4 planes. Fl. intensity is fluorescence intensity; 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: Aurora B kinase and INCENP localize to both centromeres and kinetochores in mitotic cells. (A) Immunofluorescence images of an early prometaphase control HeLa cell stained with antibodies to pABK-T232, CENP-C, and the N-terminus of Hec1 (antibody 9G3). Arrows point to the kinetochore pairs shown in the insets. A linescan through the centromere/kinetochore region is shown to the right of the panel. (B) Immunofluorescence images of an early prometaphase control HeLa cell stained with antibodies to pINCENP, CENP-C, and the N terminus of Hec1 (antibody 9G3). Numbers indicate kinetochore pairs shown in the insets, and linescans through the centromere/kinetochore region are shown to the right of each panel. pINCENP is localized to the kinetochore (pair 1), the centromere and kinetochore (pair 2), or the kinetochore-proximal outer centromere (pair 3). (C) Plots of the mean distance between the indicated proteins. Each point on the graph represents a distance measurement for a pair of sister chromatids, and the mean value is indicated. Error bars indicate SD. n values are listed in . (D) Summary of distance measurements from experiments in the study. Scale bars, 10 µm (whole cells) and 1 µm (insets). Whole cell images shown are maximum intensity projections of z-stacks containing 64 planes, and inset images shown are projections from 2 to 4 planes. Fl. intensity is fluorescence intensity; AU, arbitrary units.

Techniques: Immunofluorescence, Staining, Fluorescence

High levels of RepoMan and Aurora B predict poor outcome in cancer patients. (A) CDCA2 and AURKB expression in different cancer types and adjacent normal tissues. The box plot is based on data from TCGA and is generated using the GEPIA database. Data are presented as log2 (TPM, transcripts per million +1; * P < 0.01 using the one-way ANOVA test). BRCA, breast invasive carcinoma; KIRC, kidney renal clear cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma. (B) Scatter plot showing the Pearson correlation analysis between CDCA2 and AURKB expression in breast invasive carcinoma (TCGA, provisional). mRNA expression data (array z-score) of CDCA2 and AURKB were obtained from human cancer data sets in the cBioPortal database. r , Pearson’s correlation coefficient; P values for paired t test. (C) Correlation between CDCA2 and AURKB protein expression levels in the BRCA TCGA tumors. Protein abundances were determined by mass spectrometry (the National Cancer Institute Clinical Proteomic Tumor Analysis Consortium). r , Pearson’s correlation coefficient; P values for paired t test. (D) Representative immunostained tissue sections from normal liver tissue (RepoMan, Patient ID: 3402; Aurora B, Patient ID: 1720) and liver cholangiocarcinoma (Patient ID: 2279) in the HPA. IHC staining were performed with the antibodies HPA030049 (RepoMan) and CAB005862 (Aurora B). (E) The dot plot shows a semi-quantitative analysis of RepoMan and Aurora-B staining intensity (the values strong, moderate, weak, and negative that are used to describe intensity were transformed into 3, 2, 1, and 0, respectively) among three normal cases and ≥5 samples of liver choloangiocarcinoma from the HPA. (F) The OncoPrint from cBioPortal shows genetic alterations in CDCA2 and AURKB in 1960 (70%) out of 2815 patients with the indicated cancers. GBM, glioblastoma multiforme; PAAD, pancreatic adenocarcinoma; SKCM, skin cutaneous melanoma; SARC, sarcoma. Percentages on the right refer to genetic alterations in CDCA2 (55%) and AURKB (51%). Gain: low-level gene amplification event; amplification: high-level gene amplification event; deep deletion: homozygous (total) loss; shallow deletion: heterozygous deletion. (G) Kaplan–Meier plots comparing survival of patients with combined high and/or low expression of CDCA2 and AURKB , based on TCGA data for the indicated cancers. Survival analysis showing the effects of CDCA2 or AURKB alone for liver and lung cancer patients are shown in Supplemental Figure S3C.

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: High levels of RepoMan and Aurora B predict poor outcome in cancer patients. (A) CDCA2 and AURKB expression in different cancer types and adjacent normal tissues. The box plot is based on data from TCGA and is generated using the GEPIA database. Data are presented as log2 (TPM, transcripts per million +1; * P < 0.01 using the one-way ANOVA test). BRCA, breast invasive carcinoma; KIRC, kidney renal clear cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma. (B) Scatter plot showing the Pearson correlation analysis between CDCA2 and AURKB expression in breast invasive carcinoma (TCGA, provisional). mRNA expression data (array z-score) of CDCA2 and AURKB were obtained from human cancer data sets in the cBioPortal database. r , Pearson’s correlation coefficient; P values for paired t test. (C) Correlation between CDCA2 and AURKB protein expression levels in the BRCA TCGA tumors. Protein abundances were determined by mass spectrometry (the National Cancer Institute Clinical Proteomic Tumor Analysis Consortium). r , Pearson’s correlation coefficient; P values for paired t test. (D) Representative immunostained tissue sections from normal liver tissue (RepoMan, Patient ID: 3402; Aurora B, Patient ID: 1720) and liver cholangiocarcinoma (Patient ID: 2279) in the HPA. IHC staining were performed with the antibodies HPA030049 (RepoMan) and CAB005862 (Aurora B). (E) The dot plot shows a semi-quantitative analysis of RepoMan and Aurora-B staining intensity (the values strong, moderate, weak, and negative that are used to describe intensity were transformed into 3, 2, 1, and 0, respectively) among three normal cases and ≥5 samples of liver choloangiocarcinoma from the HPA. (F) The OncoPrint from cBioPortal shows genetic alterations in CDCA2 and AURKB in 1960 (70%) out of 2815 patients with the indicated cancers. GBM, glioblastoma multiforme; PAAD, pancreatic adenocarcinoma; SKCM, skin cutaneous melanoma; SARC, sarcoma. Percentages on the right refer to genetic alterations in CDCA2 (55%) and AURKB (51%). Gain: low-level gene amplification event; amplification: high-level gene amplification event; deep deletion: homozygous (total) loss; shallow deletion: heterozygous deletion. (G) Kaplan–Meier plots comparing survival of patients with combined high and/or low expression of CDCA2 and AURKB , based on TCGA data for the indicated cancers. Survival analysis showing the effects of CDCA2 or AURKB alone for liver and lung cancer patients are shown in Supplemental Figure S3C.

Article Snippet: Proteomic analyses of TCGA breast cancer samples also disclosed a strong positive correlation between RepoMan and Aurora-B protein levels ( ) and immunohistochemical data from the Human Protein Atlas (HPA) database showed a co–up-regulation of RepoMan and Aurora B in choloangiocarcinoma tissue sections ( ).

Techniques: Expressing, Generated, Mass Spectrometry, Immunohistochemistry, Staining, Transformation Assay, Amplification

The expression of RepoMan is cell-cycle regulated. (A) WI-38 and A549 cells were either nonsynchronized (NonSync) or arrested in prometaphase (Mitotic) by nocodazole arrest. Cell lysates were used for immunoblotting. GAPDH served as loading control (*the residual band is Aurora B after reprobe of the blot for GAPDH). (B) Representative immunofluorescence images of nonsynchronized A549 cells. The white arrow indicates an example of early prophase cell (identified by chromosome condensation within an intact nuclear envelope; Kireeva et al. , 2004 ). Scale bar, 5 µM. (C) Pearson correlation analysis of Aurora-B and RepoMan levels quantified by immunostaining in single prophase A549 cells. r , Pearson’s correlation coefficient; P values for paired t test. Each individual dot represents the signal of the mean pixel intensities of RepoMan or Aurora B normalized to DAPI (DNA). The scatter plot shows values obtained from 7–10 prophase cells from each of three independent experiments. (D) Line plot of normalized mRNA expression profiles of CDCA2 and AURKB in HeLa cells at different cell-cycle phases, as obtained from the CycleBase data set. The y -axis indicates normalized mRNA expression data plotted on time scale. The x -axis indicates different phases of the cell cycle. (E) U2OS cells were arrested at the G1/S boundary with a double-thymidine block, released into fresh medium and harvested at the indicated times. Cell lysates were analyzed by immunoblotting (*the residual band is Aurora B after reprobe of the blot for GAPDH). (F) Quantification of RepoMan and Aurora B protein abundance from four technical replicates performed as in E. RepoMan and Aurora-B band intensities were quantified and normalized to GAPDH and to 0 h. Curves indicate mean percentages ± SD. (G) Nonsynchronized U2OS cells were treated with DMSO (vehicle), cycloheximide (CHX), or MG123 and harvested at the indicated times for immunoblotting. (H) Quantification of RepoMan protein abundance from three experiments performed as in G. RepoMan band intensities were quantified and normalized to GAPDH and to the control (DMSO at 0 h) for three replicates. Curves indicate mean percentages ± SD.

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: The expression of RepoMan is cell-cycle regulated. (A) WI-38 and A549 cells were either nonsynchronized (NonSync) or arrested in prometaphase (Mitotic) by nocodazole arrest. Cell lysates were used for immunoblotting. GAPDH served as loading control (*the residual band is Aurora B after reprobe of the blot for GAPDH). (B) Representative immunofluorescence images of nonsynchronized A549 cells. The white arrow indicates an example of early prophase cell (identified by chromosome condensation within an intact nuclear envelope; Kireeva et al. , 2004 ). Scale bar, 5 µM. (C) Pearson correlation analysis of Aurora-B and RepoMan levels quantified by immunostaining in single prophase A549 cells. r , Pearson’s correlation coefficient; P values for paired t test. Each individual dot represents the signal of the mean pixel intensities of RepoMan or Aurora B normalized to DAPI (DNA). The scatter plot shows values obtained from 7–10 prophase cells from each of three independent experiments. (D) Line plot of normalized mRNA expression profiles of CDCA2 and AURKB in HeLa cells at different cell-cycle phases, as obtained from the CycleBase data set. The y -axis indicates normalized mRNA expression data plotted on time scale. The x -axis indicates different phases of the cell cycle. (E) U2OS cells were arrested at the G1/S boundary with a double-thymidine block, released into fresh medium and harvested at the indicated times. Cell lysates were analyzed by immunoblotting (*the residual band is Aurora B after reprobe of the blot for GAPDH). (F) Quantification of RepoMan and Aurora B protein abundance from four technical replicates performed as in E. RepoMan and Aurora-B band intensities were quantified and normalized to GAPDH and to 0 h. Curves indicate mean percentages ± SD. (G) Nonsynchronized U2OS cells were treated with DMSO (vehicle), cycloheximide (CHX), or MG123 and harvested at the indicated times for immunoblotting. (H) Quantification of RepoMan protein abundance from three experiments performed as in G. RepoMan band intensities were quantified and normalized to GAPDH and to the control (DMSO at 0 h) for three replicates. Curves indicate mean percentages ± SD.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Immunostaining, Blocking Assay, Quantitative Proteomics

FOXM1 controls RepoMan and Aurora-B expression. (A) Summary of the Pearson’s correlation coefficients ( r ) and P values for the indicated types of cancer, as defined in the legend of . Transcript expression data (array z-score) of CDCA2 , AURKB , and FOXM1 were obtained from human cancer data sets in the cBioPortal database. (B) U2OS cells were arrested in G2 (thymidine block and RO3306) or in mitosis (thymidine block and nocodazole) before and after the knockdown of FOXM1. Cell lysates were analyzed by immunoblotting. (C) RepoMan, Aurora-B, and FOXM1 band intensities were quantified and normalized to GAPDH and to siCTR for each phase from three independent experiments. Ns, not significant; ** P < 0.01 and *** P < 0.001 in paired t test. (D) ChIP-qPCR assay for FOXM1 of the indicated genes in U2OS cells fixed after 10 h release from a G1/S arrest. Bars indicate the mean percentages ± SD of input precipitated with FOXM1 antibody or rabbit anti-mouse immunoglobulins (IgG). ACTIN was used as non-FOXM1 target gene. ChIP enrichments were calculated as a percentage of the total input signal (Ns, not significant; * P < 0.05 in paired t test for five independent experiments). (E) Cox-proportional hazards model showing the hazard risk for the indicated variables in patients with liver hepatocellular carcinoma or lung adenocarcinoma. N, number of patients.

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: FOXM1 controls RepoMan and Aurora-B expression. (A) Summary of the Pearson’s correlation coefficients ( r ) and P values for the indicated types of cancer, as defined in the legend of . Transcript expression data (array z-score) of CDCA2 , AURKB , and FOXM1 were obtained from human cancer data sets in the cBioPortal database. (B) U2OS cells were arrested in G2 (thymidine block and RO3306) or in mitosis (thymidine block and nocodazole) before and after the knockdown of FOXM1. Cell lysates were analyzed by immunoblotting. (C) RepoMan, Aurora-B, and FOXM1 band intensities were quantified and normalized to GAPDH and to siCTR for each phase from three independent experiments. Ns, not significant; ** P < 0.01 and *** P < 0.001 in paired t test. (D) ChIP-qPCR assay for FOXM1 of the indicated genes in U2OS cells fixed after 10 h release from a G1/S arrest. Bars indicate the mean percentages ± SD of input precipitated with FOXM1 antibody or rabbit anti-mouse immunoglobulins (IgG). ACTIN was used as non-FOXM1 target gene. ChIP enrichments were calculated as a percentage of the total input signal (Ns, not significant; * P < 0.05 in paired t test for five independent experiments). (E) Cox-proportional hazards model showing the hazard risk for the indicated variables in patients with liver hepatocellular carcinoma or lung adenocarcinoma. N, number of patients.

Techniques: Expressing, Blocking Assay, Knockdown, Western Blot, ChIP-qPCR

APC/C-CDH1 targets RepoMan for proteasomal degradation at the mitotic exit. (A) Lysates and EGFP traps from nonsynchronized HEK293T cells coexpressing EGFP-tagged β-gal or EGFP-RepoMan-S893D (EGFP-RM) and either HA-CDH1 or HA-CDC20 were processed for immunoblotting. (B) In vitro ubiquitination assay of His-tagged RepoMan using purified human APC/C and CDH1. The reaction was performed for 45 min at 23°C in the absence (-) or the presence (+) of the indicated components. E1, UBE1; E2, UBCH10. RepoMan-ubiquitination was detected by immunoblotting (IB) for both RepoMan and ubiquitin, as indicated. (C) U2OS cells were transfected or not with HA-CDH1 before immunoblotting of the lysates. (D) U2OS cells were transfected for 48 h with control (siCTR) or either of two different siRNAs against CDH1 before immunoblotting of the lysates (*the residual band is Aurora B after reprobe of the blot for GAPDH). (E) Degradation curves of mClover-RepoMan after depletion of CDH1 in HeLa cells obtained by quantifying the levels of mClover-RepoMan from ≥10 cells per condition per experiment from three replicates. The intensity of fluorescence was measured at 0, 10, 20, 30, and 40 min from the beginning of metaphase, and the normalized values were plotted against time. A, anaphase; * P < 0.05 in paired t test. See also Supplemental Figure S4, E and F. (F) Immunoblot analysis of EGFP traps of lysates from nonsynchronized HEK293T cells expressing EGFP-tagged RepoMan-S893D or the indicated corresponding deletion mutants. (G) Conservation of residues 441–472 of RepoMan in mammals using Clustal Omega program formatting. (H) Effect of the deletion of residues 441–472 (Δ441–472) or alanine mutation of residues 455–457 (LEN/AAA) on the binding of CDH1 to EGFP-RepoMan-S893D in HEK293T lysates. (I) Immunoblot analysis of lysates from nonsynchronized HEK293T cells expressing EGFP-tagged RepoMan-S893D or the indicated mutants in the absence (-) or the presence (+) of HA-CDH1. (J) Relative abundance of EGFP-RepoMan levels from four independent experiments normalized to GAPDH and to the control (no transfection of HA-CDH1). Ns, not significant; * P < 0.05 in paired t test. (K) Immunoblot analysis of lysates from nonsynchronized HEK293T cells expressing EGFP-tagged RepoMan-S893D or the indicated mutants after transfection with siCTR (-) or siCDH1 (+). (L) Quantification of EGFP-RepoMan levels from three independent experiments normalized to GAPDH and to the control (siCTR). Ns, not significant; * P < 0.05 in paired t test. Since the expression level of the RepoMan (mutants) showed small differences, we compared each RepoMan variant with its own control (no HA-CDH1 or siCTR) for quantifications shown in J and L. (M) Cartoon of the APC/C associated with the cofactor CDH1 (adapted from ). RM, RepoMan; TPR, tetratricopeptide repeat; UBC, ubiquitin-conjugating enzymes.

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: APC/C-CDH1 targets RepoMan for proteasomal degradation at the mitotic exit. (A) Lysates and EGFP traps from nonsynchronized HEK293T cells coexpressing EGFP-tagged β-gal or EGFP-RepoMan-S893D (EGFP-RM) and either HA-CDH1 or HA-CDC20 were processed for immunoblotting. (B) In vitro ubiquitination assay of His-tagged RepoMan using purified human APC/C and CDH1. The reaction was performed for 45 min at 23°C in the absence (-) or the presence (+) of the indicated components. E1, UBE1; E2, UBCH10. RepoMan-ubiquitination was detected by immunoblotting (IB) for both RepoMan and ubiquitin, as indicated. (C) U2OS cells were transfected or not with HA-CDH1 before immunoblotting of the lysates. (D) U2OS cells were transfected for 48 h with control (siCTR) or either of two different siRNAs against CDH1 before immunoblotting of the lysates (*the residual band is Aurora B after reprobe of the blot for GAPDH). (E) Degradation curves of mClover-RepoMan after depletion of CDH1 in HeLa cells obtained by quantifying the levels of mClover-RepoMan from ≥10 cells per condition per experiment from three replicates. The intensity of fluorescence was measured at 0, 10, 20, 30, and 40 min from the beginning of metaphase, and the normalized values were plotted against time. A, anaphase; * P < 0.05 in paired t test. See also Supplemental Figure S4, E and F. (F) Immunoblot analysis of EGFP traps of lysates from nonsynchronized HEK293T cells expressing EGFP-tagged RepoMan-S893D or the indicated corresponding deletion mutants. (G) Conservation of residues 441–472 of RepoMan in mammals using Clustal Omega program formatting. (H) Effect of the deletion of residues 441–472 (Δ441–472) or alanine mutation of residues 455–457 (LEN/AAA) on the binding of CDH1 to EGFP-RepoMan-S893D in HEK293T lysates. (I) Immunoblot analysis of lysates from nonsynchronized HEK293T cells expressing EGFP-tagged RepoMan-S893D or the indicated mutants in the absence (-) or the presence (+) of HA-CDH1. (J) Relative abundance of EGFP-RepoMan levels from four independent experiments normalized to GAPDH and to the control (no transfection of HA-CDH1). Ns, not significant; * P < 0.05 in paired t test. (K) Immunoblot analysis of lysates from nonsynchronized HEK293T cells expressing EGFP-tagged RepoMan-S893D or the indicated mutants after transfection with siCTR (-) or siCDH1 (+). (L) Quantification of EGFP-RepoMan levels from three independent experiments normalized to GAPDH and to the control (siCTR). Ns, not significant; * P < 0.05 in paired t test. Since the expression level of the RepoMan (mutants) showed small differences, we compared each RepoMan variant with its own control (no HA-CDH1 or siCTR) for quantifications shown in J and L. (M) Cartoon of the APC/C associated with the cofactor CDH1 (adapted from ). RM, RepoMan; TPR, tetratricopeptide repeat; UBC, ubiquitin-conjugating enzymes.

Techniques: Western Blot, In Vitro, Ubiquitin Proteomics, Purification, Transfection, Control, Fluorescence, Expressing, Mutagenesis, Binding Assay, Variant Assay

The SCF FBXW7 complex promotes RepoMan degradation in interphase. (A) EGFP traps of lysates from nonsynchronized HEK293T cells coexpressing 3xFlag-FBXW7α and either EGFP-tagged β-gal or EGFP-RepoMan-S893D were processed for immunoblotting. (B) In vitro ubiquitination of His-RepoMan by recombinant SCF FBXW7 . The reaction was performed in the presence of E1 (UBE1) and E2 (UBCH3) at 30°C for 90 min. RepoMan-ubiquitination was detected by immunoblotting (IB) for both RepoMan and ubiquitin, as indicated. (C) Immunoblot analysis of lysates from U2OS cells arrested in G1/S phase nontransfected or transfected with 3xFlag-FBXW7α and siCTR (-) or 3xFlag-FBXW7α and siFBXW7 (+). *Residual band after reprobe of the blot for GAPDH. (D) Immunoblot analysis of EGFP traps from nonsynchronized HEK293T cells expressing EGFP-RepoMan-S893D and one of the indicated mutants of FBXW7α. (E) EGFP-RepoMan-S893D traps from nonsynchronized HEK293T cells were preincubated with buffer or lambda phosphatase (lambda PP) and examined for retained ectopically expressed FBXW7α. (F) Schematic representation of the predicted and established CDK phosphorylation sites of human RepoMan. Red, established phosphorylation site ( Dephoure et al. , 2008 ; Olsen et al. , 2010 ; Vagnarelli et al. , 2011 ; Prévost et al. , 2013 ; Qian et al. , 2015 ). Black, CDK phosphorylation sites, as determined by mass spectrometry ( Wu et al. , 2018 ); gray, CDK phospho-sites predicted by NetPhos 3.1 ( Blom et al. , 2004 ). (G) HEK293T cells that transiently expressed EGFP-RepoMan-S893D and 3xFlag-FBXW7α were arrested in G1/S with a single thymidine block and treated for 4 h with DMSO or 20 μM roscovitine to examine the effect on the retention of FBXW7α by EGFP-traps. (H) Effect of the deletion of residues 400–550 (∆400–550) on the binding of ectopically expressed 3x-Flag-FBXW7α and endogenous CDK2 to EGFP-RepoMan-S893D in G1/S HEK293T cells. (I) Immunoblot analysis of lysates from G1/S HEK293T cells expressing EGFP-tagged RepoMan-S893D or ∆400-550 in the absence (-) or presence (+) of 3x-Flag-FBXW7α. (J) Relative abundance of EGFP-RepoMan levels from five independent experiments normalized to GAPDH and to the control (no transfection of 3xFlag-FBXW7α). Ns, not significant; ** P value < 0.01 in paired t test. (K) Cartoon of the SCF FBXW7 complex associated with phosphorylated RepoMan (RM). Adapted from Crusio et al. (2010) . SKP1, S-phase kinase-associated protein 1; CUL1, Cullin-1; RBX1, E3 ubiquitin-protein ligase RBX1.

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: The SCF FBXW7 complex promotes RepoMan degradation in interphase. (A) EGFP traps of lysates from nonsynchronized HEK293T cells coexpressing 3xFlag-FBXW7α and either EGFP-tagged β-gal or EGFP-RepoMan-S893D were processed for immunoblotting. (B) In vitro ubiquitination of His-RepoMan by recombinant SCF FBXW7 . The reaction was performed in the presence of E1 (UBE1) and E2 (UBCH3) at 30°C for 90 min. RepoMan-ubiquitination was detected by immunoblotting (IB) for both RepoMan and ubiquitin, as indicated. (C) Immunoblot analysis of lysates from U2OS cells arrested in G1/S phase nontransfected or transfected with 3xFlag-FBXW7α and siCTR (-) or 3xFlag-FBXW7α and siFBXW7 (+). *Residual band after reprobe of the blot for GAPDH. (D) Immunoblot analysis of EGFP traps from nonsynchronized HEK293T cells expressing EGFP-RepoMan-S893D and one of the indicated mutants of FBXW7α. (E) EGFP-RepoMan-S893D traps from nonsynchronized HEK293T cells were preincubated with buffer or lambda phosphatase (lambda PP) and examined for retained ectopically expressed FBXW7α. (F) Schematic representation of the predicted and established CDK phosphorylation sites of human RepoMan. Red, established phosphorylation site ( Dephoure et al. , 2008 ; Olsen et al. , 2010 ; Vagnarelli et al. , 2011 ; Prévost et al. , 2013 ; Qian et al. , 2015 ). Black, CDK phosphorylation sites, as determined by mass spectrometry ( Wu et al. , 2018 ); gray, CDK phospho-sites predicted by NetPhos 3.1 ( Blom et al. , 2004 ). (G) HEK293T cells that transiently expressed EGFP-RepoMan-S893D and 3xFlag-FBXW7α were arrested in G1/S with a single thymidine block and treated for 4 h with DMSO or 20 μM roscovitine to examine the effect on the retention of FBXW7α by EGFP-traps. (H) Effect of the deletion of residues 400–550 (∆400–550) on the binding of ectopically expressed 3x-Flag-FBXW7α and endogenous CDK2 to EGFP-RepoMan-S893D in G1/S HEK293T cells. (I) Immunoblot analysis of lysates from G1/S HEK293T cells expressing EGFP-tagged RepoMan-S893D or ∆400-550 in the absence (-) or presence (+) of 3x-Flag-FBXW7α. (J) Relative abundance of EGFP-RepoMan levels from five independent experiments normalized to GAPDH and to the control (no transfection of 3xFlag-FBXW7α). Ns, not significant; ** P value < 0.01 in paired t test. (K) Cartoon of the SCF FBXW7 complex associated with phosphorylated RepoMan (RM). Adapted from Crusio et al. (2010) . SKP1, S-phase kinase-associated protein 1; CUL1, Cullin-1; RBX1, E3 ubiquitin-protein ligase RBX1.

Techniques: Western Blot, In Vitro, Ubiquitin Proteomics, Recombinant, Transfection, Expressing, Phospho-proteomics, Mass Spectrometry, Blocking Assay, Binding Assay, Control

A theoretical model captures the co-regulation of RepoMan and Aurora B. (A) Diagram showing protein interactions in a simplified cell-cycle regulatory network. (B) The CDK1 module generates a bistable switch in steady state. For low and high levels of Cyclin B, only the corresponding low or high activities of CDK1 exists, but for intermediate values (shaded zone), CDK1 can be either in a state of low activity or high activity. (C) The Aurora-B module can also generate a bistable switch of Aurora-B activity in response to CDK1 activity. The shape of the curve is modified by changing abundances of RepoMan and Aurora-B. (D) Time series simulation of the coupled modules in A during one cell cycle. The timing is determined by production and degradation of the different proteins via ubiquitination by ligases such as APC/C and SCF FBXW7 . First row: levels of Aurora-B abundance, CDK1 abundance, and Cyclin B abundance over time. Second row: response of Aurora-B activity to CDK1 activity (dashed line). The blue line traces the current levels of CDK1 activity and Aurora-B activity (red dot). In particular, it shows how the activities change differently when CDK1 activity increases and decreases. Note that, due to varying levels of total Aurora B, the response curve also changes. Third row: response of CDK1 activity to Cyclin B abundance (dashed curve) and current levels (red dot). Since total CDK1 remains constant, the response curve does not change in time. (E) Large or small amplitude oscillations in Aurora-B activity (see profiles 1-2-3) can be obtained in response to periodic changes (with varying mean and amplitude) of the total abundance of both Aurora B and RepoMan. For details, see Materials and Methods and Supplemental Figure S6. (F) By increasing RepoMan abundance, the sensitivity of Aurora B to inhibitors is increased.

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: A theoretical model captures the co-regulation of RepoMan and Aurora B. (A) Diagram showing protein interactions in a simplified cell-cycle regulatory network. (B) The CDK1 module generates a bistable switch in steady state. For low and high levels of Cyclin B, only the corresponding low or high activities of CDK1 exists, but for intermediate values (shaded zone), CDK1 can be either in a state of low activity or high activity. (C) The Aurora-B module can also generate a bistable switch of Aurora-B activity in response to CDK1 activity. The shape of the curve is modified by changing abundances of RepoMan and Aurora-B. (D) Time series simulation of the coupled modules in A during one cell cycle. The timing is determined by production and degradation of the different proteins via ubiquitination by ligases such as APC/C and SCF FBXW7 . First row: levels of Aurora-B abundance, CDK1 abundance, and Cyclin B abundance over time. Second row: response of Aurora-B activity to CDK1 activity (dashed line). The blue line traces the current levels of CDK1 activity and Aurora-B activity (red dot). In particular, it shows how the activities change differently when CDK1 activity increases and decreases. Note that, due to varying levels of total Aurora B, the response curve also changes. Third row: response of CDK1 activity to Cyclin B abundance (dashed curve) and current levels (red dot). Since total CDK1 remains constant, the response curve does not change in time. (E) Large or small amplitude oscillations in Aurora-B activity (see profiles 1-2-3) can be obtained in response to periodic changes (with varying mean and amplitude) of the total abundance of both Aurora B and RepoMan. For details, see Materials and Methods and Supplemental Figure S6. (F) By increasing RepoMan abundance, the sensitivity of Aurora B to inhibitors is increased.

Techniques: Activity Assay, Modification, Ubiquitin Proteomics

Overexpression of RepoMan sensitizes cancer cells to Aurora-B inhibitors. (A) Monastrol-arrested HeLa Flp-In T-REx cells were treated for 1 h with the indicated concentrations of hesperadin before (-DOX) and after induction (+DOX) of mClover-tagged RepoMan. Cells were fixed and stained. (B) Quantification of the H3S10ph/DNA ratio in A. The graph shows the mean percentage ± SD from four independent experiments (≥25 cells for each condition per experiment). * P < 0.05 in paired t test. (C) Percentage of confluence over time of HeLa Flp-In T-REx cells before and after induction with Dox and treated with either DMSO or 20 nM hesperadin. The growth curves are representative of three experiments and were obtained from confluence measurements acquired at 2 h intervals using IncuCyte software. (D) Same as C but after treatment with DMSO or 15 nM AZD1152. (E) ZM447439 sensitivity (IC50) prediction from cancer cell lines (CCLE) when comparing low and high AURKB or CDCA2 expression. Differences in median log(IC50) across the subgroups were evaluated with the Wilcoxon–Mann–Whitney Test. (F) The co–up-regulation of CDCA2 and AURKB (high_high) significantly reduces the log(IC50) of ZM447439 in cancer cell lines (CCLE). Differences in median log(IC50) across the subgroups were evaluated with the Wilcoxon–Mann–Whitney Test.

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: Overexpression of RepoMan sensitizes cancer cells to Aurora-B inhibitors. (A) Monastrol-arrested HeLa Flp-In T-REx cells were treated for 1 h with the indicated concentrations of hesperadin before (-DOX) and after induction (+DOX) of mClover-tagged RepoMan. Cells were fixed and stained. (B) Quantification of the H3S10ph/DNA ratio in A. The graph shows the mean percentage ± SD from four independent experiments (≥25 cells for each condition per experiment). * P < 0.05 in paired t test. (C) Percentage of confluence over time of HeLa Flp-In T-REx cells before and after induction with Dox and treated with either DMSO or 20 nM hesperadin. The growth curves are representative of three experiments and were obtained from confluence measurements acquired at 2 h intervals using IncuCyte software. (D) Same as C but after treatment with DMSO or 15 nM AZD1152. (E) ZM447439 sensitivity (IC50) prediction from cancer cell lines (CCLE) when comparing low and high AURKB or CDCA2 expression. Differences in median log(IC50) across the subgroups were evaluated with the Wilcoxon–Mann–Whitney Test. (F) The co–up-regulation of CDCA2 and AURKB (high_high) significantly reduces the log(IC50) of ZM447439 in cancer cell lines (CCLE). Differences in median log(IC50) across the subgroups were evaluated with the Wilcoxon–Mann–Whitney Test.

Techniques: Over Expression, Staining, Software, Expressing, MANN-WHITNEY

Model for the co-regulation of RepoMan and Aurora B in cancer cells. (A) RepoMan and Aurora B are co-regulated at multiple levels during the cell cycle. Gray zone: the two counteracting enzymes have maximal expression in G2/M in a FOXM1-dependent manner; white zone: during mitotic exit and early G1 the ubiquitin ligase APC/C-CDH1 down-regulates RepoMan and Aurora B; during interphase (likely G1/S transition) SCF FBXW7 is involved in the proteolytic turnover of RepoMan and Aurora B; CDCA2 and AURKB transcripts are down-regulated in S phase by a mRNA decay pathway involving ERG-CCR4-NOT. (B) Phenomenological model that captures how the growth rate of cancer cells changes with varying concentrations of RepoMan and Aurora B, as well as their ratio. (C) Hypothetical model for RepoMan and Aurora-B co–up-regulation in cancer cells based on B. Cancer cells (gray ovals) with a low and balanced ratio of RepoMan and Aurora B (black scale) grow slowly (light green bar), as compared with tumor cells with high and balanced RepoMan:Aurora-B ratio (red scale and dark green bar). Unbalanced levels of the two proteins (gray scale) is disadvantageous for cell proliferation (pale green bar).

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.E19-12-0698

Figure Lengend Snippet: Model for the co-regulation of RepoMan and Aurora B in cancer cells. (A) RepoMan and Aurora B are co-regulated at multiple levels during the cell cycle. Gray zone: the two counteracting enzymes have maximal expression in G2/M in a FOXM1-dependent manner; white zone: during mitotic exit and early G1 the ubiquitin ligase APC/C-CDH1 down-regulates RepoMan and Aurora B; during interphase (likely G1/S transition) SCF FBXW7 is involved in the proteolytic turnover of RepoMan and Aurora B; CDCA2 and AURKB transcripts are down-regulated in S phase by a mRNA decay pathway involving ERG-CCR4-NOT. (B) Phenomenological model that captures how the growth rate of cancer cells changes with varying concentrations of RepoMan and Aurora B, as well as their ratio. (C) Hypothetical model for RepoMan and Aurora-B co–up-regulation in cancer cells based on B. Cancer cells (gray ovals) with a low and balanced ratio of RepoMan and Aurora B (black scale) grow slowly (light green bar), as compared with tumor cells with high and balanced RepoMan:Aurora-B ratio (red scale and dark green bar). Unbalanced levels of the two proteins (gray scale) is disadvantageous for cell proliferation (pale green bar).

Techniques: Expressing, Ubiquitin Proteomics

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