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crest antibody human cs1058  (Cortex Biochem Inc)

 
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    Cortex Biochem Inc crest antibody human cs1058
    CENP-A and <t>CREST</t> colocalize regardless of ICRF-193 treatment in HeLa cells. (A) Cartoon depicts centromere/kinetochore regions. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; CENP-A, green. Bars, 10 µm (insets, 1 µm). (C) Quantification of <t>immunofluorescent</t> <t>staining</t> at centromeres (Cen). Error bars, standard deviation. (D) Distribution plot of KPC-to-KPC distances. Error bars, standard deviation. Data were collected from three independent experiments.
    Crest Antibody Human Cs1058, supplied by Cortex Biochem Inc, 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/crest antibody human cs1058/product/Cortex Biochem Inc
    Average 90 stars, based on 1 article reviews
    crest antibody human cs1058 - by Bioz Stars, 2026-03
    90/100 stars

    Images

    1) Product Images from "Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases"

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201807189

    CENP-A and CREST colocalize regardless of ICRF-193 treatment in HeLa cells. (A) Cartoon depicts centromere/kinetochore regions. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; CENP-A, green. Bars, 10 µm (insets, 1 µm). (C) Quantification of immunofluorescent staining at centromeres (Cen). Error bars, standard deviation. (D) Distribution plot of KPC-to-KPC distances. Error bars, standard deviation. Data were collected from three independent experiments.
    Figure Legend Snippet: CENP-A and CREST colocalize regardless of ICRF-193 treatment in HeLa cells. (A) Cartoon depicts centromere/kinetochore regions. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; CENP-A, green. Bars, 10 µm (insets, 1 µm). (C) Quantification of immunofluorescent staining at centromeres (Cen). Error bars, standard deviation. (D) Distribution plot of KPC-to-KPC distances. Error bars, standard deviation. Data were collected from three independent experiments.

    Techniques Used: Staining, Standard Deviation

    Aurora B recruitment to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; Aurora B, green. Line scans were done in the x and y axis. Bars, 10 µm (insets, 1 µm). (B) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. (C) Averaged plots of Aurora B and CREST signal intensities in line scans across centromeres in cells treated as in A. Each scan was normalized to the highest value. Control, n = 52 chromosomes from 25 cells; ICRF, n = 50 chromosomes from 31 cells. (D) Images showing line scans of Aurora B and CENP-A perpendicular to the kinetochore–kinetochore axis in metaphase cells (no nocodazole). Bars, 1 µm. (E) Quantification of peak CENP-A signals from D. Control, n = 15 cells and n = 124 kinetochores; ICRF-193, n = 12 cells and n = 44 kinetochores; P = 0.85. Data collected from three independent experiments. P values from Student’s t test. n.s., not significant. (F) Quantification of Aurora B/CENP-A ratios from D. Control, n = 40 cells and n = 204 kinetochores; ICRF-193, n = 42 cells and n = 172 kinetochores; ****, P = 2 × 10 −39 . Data collected from at least three independent experiments. P values from Student’s t test. All error bars, standard deviation.
    Figure Legend Snippet: Aurora B recruitment to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; Aurora B, green. Line scans were done in the x and y axis. Bars, 10 µm (insets, 1 µm). (B) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. (C) Averaged plots of Aurora B and CREST signal intensities in line scans across centromeres in cells treated as in A. Each scan was normalized to the highest value. Control, n = 52 chromosomes from 25 cells; ICRF, n = 50 chromosomes from 31 cells. (D) Images showing line scans of Aurora B and CENP-A perpendicular to the kinetochore–kinetochore axis in metaphase cells (no nocodazole). Bars, 1 µm. (E) Quantification of peak CENP-A signals from D. Control, n = 15 cells and n = 124 kinetochores; ICRF-193, n = 12 cells and n = 44 kinetochores; P = 0.85. Data collected from three independent experiments. P values from Student’s t test. n.s., not significant. (F) Quantification of Aurora B/CENP-A ratios from D. Control, n = 40 cells and n = 204 kinetochores; ICRF-193, n = 42 cells and n = 172 kinetochores; ****, P = 2 × 10 −39 . Data collected from at least three independent experiments. P values from Student’s t test. All error bars, standard deviation.

    Techniques Used: Staining, Standard Deviation, Control

    Topo II inhibitors etoposide and merbarone only weakly affect Aurora B localization in mitotic chromosomes. (A and B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) after merbarone (200 µM) or etoposide (10 µM) treatment for 45 min. Bars, 10 µm (insets, 1 µm). CREST, red; Aurora B, green. (C) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Etoposide, n = 873 chromosomes; merbarone, n = 683 chromosomes. Data were collected from at least three independent experiments. (D) Representative examples of live-cell imaging showing normal anaphase onset, metaphase arrest, and decondensation (Decon) of chromatin following attempted anaphase when Topo II is inhibited and chromosomes cannot segregate. Bars, 10 µm.
    Figure Legend Snippet: Topo II inhibitors etoposide and merbarone only weakly affect Aurora B localization in mitotic chromosomes. (A and B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) after merbarone (200 µM) or etoposide (10 µM) treatment for 45 min. Bars, 10 µm (insets, 1 µm). CREST, red; Aurora B, green. (C) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Etoposide, n = 873 chromosomes; merbarone, n = 683 chromosomes. Data were collected from at least three independent experiments. (D) Representative examples of live-cell imaging showing normal anaphase onset, metaphase arrest, and decondensation (Decon) of chromatin following attempted anaphase when Topo II is inhibited and chromosomes cannot segregate. Bars, 10 µm.

    Techniques Used: Staining, Standard Deviation, Live Cell Imaging

    Aurora B or Haspin inhibition bypasses the metaphase checkpoint induced by Topo II catalytic inhibitors. (A) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Hesperadin (1 µM), an Aurora B inhibitor (Hauf et al., 2003), and 5-ITu (10 µM), a Haspin inhibitor (De Antoni et al., 2012), were added to metaphase cells 10 min after ICRF-193 was added. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. Cells that remained arrested in metaphase for the duration of the experiment are indicated by a red bar. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) treated with ICRF-193 ± 5-ITu for 45 min. CREST, red; Aurora B, green. After ICRF-193 treatment, Aurora B localizes to the KPCs and chromosome core (right). After ICRF-193 + 5-ITu treatment (left), Aurora B typically localizes diffusely on chromatin and is present on some KPCs. The histogram plot shows classification of Aurora B staining pattern. Error bars, standard deviation. Bars, 10 µm. Data were collected from at least three independent experiments.
    Figure Legend Snippet: Aurora B or Haspin inhibition bypasses the metaphase checkpoint induced by Topo II catalytic inhibitors. (A) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Hesperadin (1 µM), an Aurora B inhibitor (Hauf et al., 2003), and 5-ITu (10 µM), a Haspin inhibitor (De Antoni et al., 2012), were added to metaphase cells 10 min after ICRF-193 was added. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. Cells that remained arrested in metaphase for the duration of the experiment are indicated by a red bar. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) treated with ICRF-193 ± 5-ITu for 45 min. CREST, red; Aurora B, green. After ICRF-193 treatment, Aurora B localizes to the KPCs and chromosome core (right). After ICRF-193 + 5-ITu treatment (left), Aurora B typically localizes diffusely on chromatin and is present on some KPCs. The histogram plot shows classification of Aurora B staining pattern. Error bars, standard deviation. Bars, 10 µm. Data were collected from at least three independent experiments.

    Techniques Used: Inhibition, Single-cell Analysis, Quantitation Assay, Staining, Standard Deviation

    INCENP and H3T3p at KPCs and chromosome arms after ICRF-193 treatment in mitosis. (A and C) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. Bars, 10 µm (insets, 1 µm). (A) CREST, red; INCENP, green. (C) CREST, red. H3T3p, green. (B and D) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Data collected from three independent experiments.
    Figure Legend Snippet: INCENP and H3T3p at KPCs and chromosome arms after ICRF-193 treatment in mitosis. (A and C) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. Bars, 10 µm (insets, 1 µm). (A) CREST, red; INCENP, green. (C) CREST, red. H3T3p, green. (B and D) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Data collected from three independent experiments.

    Techniques Used: Staining, Standard Deviation

    Haspin promotes Aurora B localization to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 ± CHR-6494 treatment for 45 min. CREST, red; Aurora B, green. (B) Classification of Aurora B staining pattern. (C) Quantification of average Aurora B signal intensity spanning chromosome arms. Each scan was normalized to the highest value. ICRF, n = 42 chromosomes from 35 cells; Haspin, n = 51 chromosomes from 27 cells. (D) Quantification of Aurora B signal intensity on chromatin. P values from Student’s t test. *, P = 0.04; **, P = 0.01; ***, P = 0.001; ****, P = 0.0004. a.u., arbitrary unit. (E) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. (F) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min. CREST, red; SUMO2/3, green. The individual channels are provided for reference in . The plot shows classification of SUMO2/3 staining pattern. Bars, 10 µm. Error bars, standard deviation. The images are representative examples of those collected from three independent experiments.
    Figure Legend Snippet: Haspin promotes Aurora B localization to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 ± CHR-6494 treatment for 45 min. CREST, red; Aurora B, green. (B) Classification of Aurora B staining pattern. (C) Quantification of average Aurora B signal intensity spanning chromosome arms. Each scan was normalized to the highest value. ICRF, n = 42 chromosomes from 35 cells; Haspin, n = 51 chromosomes from 27 cells. (D) Quantification of Aurora B signal intensity on chromatin. P values from Student’s t test. *, P = 0.04; **, P = 0.01; ***, P = 0.001; ****, P = 0.0004. a.u., arbitrary unit. (E) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. (F) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min. CREST, red; SUMO2/3, green. The individual channels are provided for reference in . The plot shows classification of SUMO2/3 staining pattern. Bars, 10 µm. Error bars, standard deviation. The images are representative examples of those collected from three independent experiments.

    Techniques Used: Staining, Single-cell Analysis, Quantitation Assay, Standard Deviation

    Topo II inhibitor ICRF-193 up-regulates Topo IIα SUMOylation and induces recruitment of SUMO2/3 to KPCs and chromosome arms in HeLa cells. (A) Representative images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min and then immunostained with CREST serum and anti-SUMO2/3 antibody. DAPI stain for DNA. These are individual channels from . Bars, 10 µm. The images are representative examples of those collected from at least three independent experiments. (B) DMSO-, ICRF-193–, and merbarone-treated mitotic chromosomes were isolated from HeLa cells and subjected to Western blotting. The mitotic SUMOylation and Topo SUMOylation were probed using the indicated antibodies. Histone H3 was probed as a loading control for the mitotic chromosomes. Percentage SUMOylation of Topo IIα was calculated from three independent experiments ( n = 3). Error bars, standard deviation. *, P value from Student’s t test. **, Statistically significant difference, P ≤ 0.01.
    Figure Legend Snippet: Topo II inhibitor ICRF-193 up-regulates Topo IIα SUMOylation and induces recruitment of SUMO2/3 to KPCs and chromosome arms in HeLa cells. (A) Representative images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min and then immunostained with CREST serum and anti-SUMO2/3 antibody. DAPI stain for DNA. These are individual channels from . Bars, 10 µm. The images are representative examples of those collected from at least three independent experiments. (B) DMSO-, ICRF-193–, and merbarone-treated mitotic chromosomes were isolated from HeLa cells and subjected to Western blotting. The mitotic SUMOylation and Topo SUMOylation were probed using the indicated antibodies. Histone H3 was probed as a loading control for the mitotic chromosomes. Percentage SUMOylation of Topo IIα was calculated from three independent experiments ( n = 3). Error bars, standard deviation. *, P value from Student’s t test. **, Statistically significant difference, P ≤ 0.01.

    Techniques Used: Staining, Isolation, Western Blot, Control, Standard Deviation



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    crest antibody human cs1058  (Cortex Biochem Inc)
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    Cortex Biochem Inc crest antibody human cs1058
    CENP-A and <t>CREST</t> colocalize regardless of ICRF-193 treatment in HeLa cells. (A) Cartoon depicts centromere/kinetochore regions. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; CENP-A, green. Bars, 10 µm (insets, 1 µm). (C) Quantification of <t>immunofluorescent</t> <t>staining</t> at centromeres (Cen). Error bars, standard deviation. (D) Distribution plot of KPC-to-KPC distances. Error bars, standard deviation. Data were collected from three independent experiments.
    Crest Antibody Human Cs1058, supplied by Cortex Biochem Inc, 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/crest antibody human cs1058/product/Cortex Biochem Inc
    Average 90 stars, based on 1 article reviews
    crest antibody human cs1058 - by Bioz Stars, 2026-03
    90/100 stars
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    CENP-A and CREST colocalize regardless of ICRF-193 treatment in HeLa cells. (A) Cartoon depicts centromere/kinetochore regions. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; CENP-A, green. Bars, 10 µm (insets, 1 µm). (C) Quantification of immunofluorescent staining at centromeres (Cen). Error bars, standard deviation. (D) Distribution plot of KPC-to-KPC distances. Error bars, standard deviation. Data were collected from three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    doi: 10.1083/jcb.201807189

    Figure Lengend Snippet: CENP-A and CREST colocalize regardless of ICRF-193 treatment in HeLa cells. (A) Cartoon depicts centromere/kinetochore regions. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; CENP-A, green. Bars, 10 µm (insets, 1 µm). (C) Quantification of immunofluorescent staining at centromeres (Cen). Error bars, standard deviation. (D) Distribution plot of KPC-to-KPC distances. Error bars, standard deviation. Data were collected from three independent experiments.

    Article Snippet: The following primary antibodies were used for staining: CREST antibody (human, CS1058, 1:200; Cortex Biochem), anti-Aurora B antibody (mouse, 611083, 1:200; BD Transduction), anti–CENP-A antibody (rabbit, 2186S, 1:200; Cell Signaling Technology), anti-INCENP antibody (rabbit, I5283, 1:2,000; Sigma-Aldrich), and anti-H3T3-Phos antibody (rabbit, ab78351, 1:1,000; Abcam).

    Techniques: Staining, Standard Deviation

    Aurora B recruitment to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; Aurora B, green. Line scans were done in the x and y axis. Bars, 10 µm (insets, 1 µm). (B) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. (C) Averaged plots of Aurora B and CREST signal intensities in line scans across centromeres in cells treated as in A. Each scan was normalized to the highest value. Control, n = 52 chromosomes from 25 cells; ICRF, n = 50 chromosomes from 31 cells. (D) Images showing line scans of Aurora B and CENP-A perpendicular to the kinetochore–kinetochore axis in metaphase cells (no nocodazole). Bars, 1 µm. (E) Quantification of peak CENP-A signals from D. Control, n = 15 cells and n = 124 kinetochores; ICRF-193, n = 12 cells and n = 44 kinetochores; P = 0.85. Data collected from three independent experiments. P values from Student’s t test. n.s., not significant. (F) Quantification of Aurora B/CENP-A ratios from D. Control, n = 40 cells and n = 204 kinetochores; ICRF-193, n = 42 cells and n = 172 kinetochores; ****, P = 2 × 10 −39 . Data collected from at least three independent experiments. P values from Student’s t test. All error bars, standard deviation.

    Journal: The Journal of Cell Biology

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    doi: 10.1083/jcb.201807189

    Figure Lengend Snippet: Aurora B recruitment to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. CREST, red; Aurora B, green. Line scans were done in the x and y axis. Bars, 10 µm (insets, 1 µm). (B) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. (C) Averaged plots of Aurora B and CREST signal intensities in line scans across centromeres in cells treated as in A. Each scan was normalized to the highest value. Control, n = 52 chromosomes from 25 cells; ICRF, n = 50 chromosomes from 31 cells. (D) Images showing line scans of Aurora B and CENP-A perpendicular to the kinetochore–kinetochore axis in metaphase cells (no nocodazole). Bars, 1 µm. (E) Quantification of peak CENP-A signals from D. Control, n = 15 cells and n = 124 kinetochores; ICRF-193, n = 12 cells and n = 44 kinetochores; P = 0.85. Data collected from three independent experiments. P values from Student’s t test. n.s., not significant. (F) Quantification of Aurora B/CENP-A ratios from D. Control, n = 40 cells and n = 204 kinetochores; ICRF-193, n = 42 cells and n = 172 kinetochores; ****, P = 2 × 10 −39 . Data collected from at least three independent experiments. P values from Student’s t test. All error bars, standard deviation.

    Article Snippet: The following primary antibodies were used for staining: CREST antibody (human, CS1058, 1:200; Cortex Biochem), anti-Aurora B antibody (mouse, 611083, 1:200; BD Transduction), anti–CENP-A antibody (rabbit, 2186S, 1:200; Cell Signaling Technology), anti-INCENP antibody (rabbit, I5283, 1:2,000; Sigma-Aldrich), and anti-H3T3-Phos antibody (rabbit, ab78351, 1:1,000; Abcam).

    Techniques: Staining, Standard Deviation, Control

    Topo II inhibitors etoposide and merbarone only weakly affect Aurora B localization in mitotic chromosomes. (A and B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) after merbarone (200 µM) or etoposide (10 µM) treatment for 45 min. Bars, 10 µm (insets, 1 µm). CREST, red; Aurora B, green. (C) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Etoposide, n = 873 chromosomes; merbarone, n = 683 chromosomes. Data were collected from at least three independent experiments. (D) Representative examples of live-cell imaging showing normal anaphase onset, metaphase arrest, and decondensation (Decon) of chromatin following attempted anaphase when Topo II is inhibited and chromosomes cannot segregate. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    doi: 10.1083/jcb.201807189

    Figure Lengend Snippet: Topo II inhibitors etoposide and merbarone only weakly affect Aurora B localization in mitotic chromosomes. (A and B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) after merbarone (200 µM) or etoposide (10 µM) treatment for 45 min. Bars, 10 µm (insets, 1 µm). CREST, red; Aurora B, green. (C) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Etoposide, n = 873 chromosomes; merbarone, n = 683 chromosomes. Data were collected from at least three independent experiments. (D) Representative examples of live-cell imaging showing normal anaphase onset, metaphase arrest, and decondensation (Decon) of chromatin following attempted anaphase when Topo II is inhibited and chromosomes cannot segregate. Bars, 10 µm.

    Article Snippet: The following primary antibodies were used for staining: CREST antibody (human, CS1058, 1:200; Cortex Biochem), anti-Aurora B antibody (mouse, 611083, 1:200; BD Transduction), anti–CENP-A antibody (rabbit, 2186S, 1:200; Cell Signaling Technology), anti-INCENP antibody (rabbit, I5283, 1:2,000; Sigma-Aldrich), and anti-H3T3-Phos antibody (rabbit, ab78351, 1:1,000; Abcam).

    Techniques: Staining, Standard Deviation, Live Cell Imaging

    Aurora B or Haspin inhibition bypasses the metaphase checkpoint induced by Topo II catalytic inhibitors. (A) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Hesperadin (1 µM), an Aurora B inhibitor (Hauf et al., 2003), and 5-ITu (10 µM), a Haspin inhibitor (De Antoni et al., 2012), were added to metaphase cells 10 min after ICRF-193 was added. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. Cells that remained arrested in metaphase for the duration of the experiment are indicated by a red bar. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) treated with ICRF-193 ± 5-ITu for 45 min. CREST, red; Aurora B, green. After ICRF-193 treatment, Aurora B localizes to the KPCs and chromosome core (right). After ICRF-193 + 5-ITu treatment (left), Aurora B typically localizes diffusely on chromatin and is present on some KPCs. The histogram plot shows classification of Aurora B staining pattern. Error bars, standard deviation. Bars, 10 µm. Data were collected from at least three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    doi: 10.1083/jcb.201807189

    Figure Lengend Snippet: Aurora B or Haspin inhibition bypasses the metaphase checkpoint induced by Topo II catalytic inhibitors. (A) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Hesperadin (1 µM), an Aurora B inhibitor (Hauf et al., 2003), and 5-ITu (10 µM), a Haspin inhibitor (De Antoni et al., 2012), were added to metaphase cells 10 min after ICRF-193 was added. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. Cells that remained arrested in metaphase for the duration of the experiment are indicated by a red bar. (B) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) treated with ICRF-193 ± 5-ITu for 45 min. CREST, red; Aurora B, green. After ICRF-193 treatment, Aurora B localizes to the KPCs and chromosome core (right). After ICRF-193 + 5-ITu treatment (left), Aurora B typically localizes diffusely on chromatin and is present on some KPCs. The histogram plot shows classification of Aurora B staining pattern. Error bars, standard deviation. Bars, 10 µm. Data were collected from at least three independent experiments.

    Article Snippet: The following primary antibodies were used for staining: CREST antibody (human, CS1058, 1:200; Cortex Biochem), anti-Aurora B antibody (mouse, 611083, 1:200; BD Transduction), anti–CENP-A antibody (rabbit, 2186S, 1:200; Cell Signaling Technology), anti-INCENP antibody (rabbit, I5283, 1:2,000; Sigma-Aldrich), and anti-H3T3-Phos antibody (rabbit, ab78351, 1:1,000; Abcam).

    Techniques: Inhibition, Single-cell Analysis, Quantitation Assay, Staining, Standard Deviation

    INCENP and H3T3p at KPCs and chromosome arms after ICRF-193 treatment in mitosis. (A and C) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. Bars, 10 µm (insets, 1 µm). (A) CREST, red; INCENP, green. (C) CREST, red. H3T3p, green. (B and D) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Data collected from three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    doi: 10.1083/jcb.201807189

    Figure Lengend Snippet: INCENP and H3T3p at KPCs and chromosome arms after ICRF-193 treatment in mitosis. (A and C) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 treatment for 45 min. Bars, 10 µm (insets, 1 µm). (A) CREST, red; INCENP, green. (C) CREST, red. H3T3p, green. (B and D) Quantification of immunofluorescent staining at centromeres/chromosome arms. Error bars, standard deviation. Data collected from three independent experiments.

    Article Snippet: The following primary antibodies were used for staining: CREST antibody (human, CS1058, 1:200; Cortex Biochem), anti-Aurora B antibody (mouse, 611083, 1:200; BD Transduction), anti–CENP-A antibody (rabbit, 2186S, 1:200; Cell Signaling Technology), anti-INCENP antibody (rabbit, I5283, 1:2,000; Sigma-Aldrich), and anti-H3T3-Phos antibody (rabbit, ab78351, 1:1,000; Abcam).

    Techniques: Staining, Standard Deviation

    Haspin promotes Aurora B localization to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 ± CHR-6494 treatment for 45 min. CREST, red; Aurora B, green. (B) Classification of Aurora B staining pattern. (C) Quantification of average Aurora B signal intensity spanning chromosome arms. Each scan was normalized to the highest value. ICRF, n = 42 chromosomes from 35 cells; Haspin, n = 51 chromosomes from 27 cells. (D) Quantification of Aurora B signal intensity on chromatin. P values from Student’s t test. *, P = 0.04; **, P = 0.01; ***, P = 0.001; ****, P = 0.0004. a.u., arbitrary unit. (E) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. (F) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min. CREST, red; SUMO2/3, green. The individual channels are provided for reference in . The plot shows classification of SUMO2/3 staining pattern. Bars, 10 µm. Error bars, standard deviation. The images are representative examples of those collected from three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    doi: 10.1083/jcb.201807189

    Figure Lengend Snippet: Haspin promotes Aurora B localization to KPCs and chromosome arms during ICRF-193 treatment in HeLa cells. (A) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 ± CHR-6494 treatment for 45 min. CREST, red; Aurora B, green. (B) Classification of Aurora B staining pattern. (C) Quantification of average Aurora B signal intensity spanning chromosome arms. Each scan was normalized to the highest value. ICRF, n = 42 chromosomes from 35 cells; Haspin, n = 51 chromosomes from 27 cells. (D) Quantification of Aurora B signal intensity on chromatin. P values from Student’s t test. *, P = 0.04; **, P = 0.01; ***, P = 0.001; ****, P = 0.0004. a.u., arbitrary unit. (E) Live single-cell analysis of mitotic progression. Quantitation of time to anaphase and decondensation during drug treatments. Each vertical bar represents one cell, with each cell being scored for time to anaphase and decondensation from the start of the time course. (F) Representative immunofluorescent-stained images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min. CREST, red; SUMO2/3, green. The individual channels are provided for reference in . The plot shows classification of SUMO2/3 staining pattern. Bars, 10 µm. Error bars, standard deviation. The images are representative examples of those collected from three independent experiments.

    Article Snippet: The following primary antibodies were used for staining: CREST antibody (human, CS1058, 1:200; Cortex Biochem), anti-Aurora B antibody (mouse, 611083, 1:200; BD Transduction), anti–CENP-A antibody (rabbit, 2186S, 1:200; Cell Signaling Technology), anti-INCENP antibody (rabbit, I5283, 1:2,000; Sigma-Aldrich), and anti-H3T3-Phos antibody (rabbit, ab78351, 1:1,000; Abcam).

    Techniques: Staining, Single-cell Analysis, Quantitation Assay, Standard Deviation

    Topo II inhibitor ICRF-193 up-regulates Topo IIα SUMOylation and induces recruitment of SUMO2/3 to KPCs and chromosome arms in HeLa cells. (A) Representative images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min and then immunostained with CREST serum and anti-SUMO2/3 antibody. DAPI stain for DNA. These are individual channels from . Bars, 10 µm. The images are representative examples of those collected from at least three independent experiments. (B) DMSO-, ICRF-193–, and merbarone-treated mitotic chromosomes were isolated from HeLa cells and subjected to Western blotting. The mitotic SUMOylation and Topo SUMOylation were probed using the indicated antibodies. Histone H3 was probed as a loading control for the mitotic chromosomes. Percentage SUMOylation of Topo IIα was calculated from three independent experiments ( n = 3). Error bars, standard deviation. *, P value from Student’s t test. **, Statistically significant difference, P ≤ 0.01.

    Journal: The Journal of Cell Biology

    Article Title: Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases

    doi: 10.1083/jcb.201807189

    Figure Lengend Snippet: Topo II inhibitor ICRF-193 up-regulates Topo IIα SUMOylation and induces recruitment of SUMO2/3 to KPCs and chromosome arms in HeLa cells. (A) Representative images of pseudometaphase HeLa cells (nocodazole arrested) ± ICRF-193 for 45 min and then immunostained with CREST serum and anti-SUMO2/3 antibody. DAPI stain for DNA. These are individual channels from . Bars, 10 µm. The images are representative examples of those collected from at least three independent experiments. (B) DMSO-, ICRF-193–, and merbarone-treated mitotic chromosomes were isolated from HeLa cells and subjected to Western blotting. The mitotic SUMOylation and Topo SUMOylation were probed using the indicated antibodies. Histone H3 was probed as a loading control for the mitotic chromosomes. Percentage SUMOylation of Topo IIα was calculated from three independent experiments ( n = 3). Error bars, standard deviation. *, P value from Student’s t test. **, Statistically significant difference, P ≤ 0.01.

    Article Snippet: The following primary antibodies were used for staining: CREST antibody (human, CS1058, 1:200; Cortex Biochem), anti-Aurora B antibody (mouse, 611083, 1:200; BD Transduction), anti–CENP-A antibody (rabbit, 2186S, 1:200; Cell Signaling Technology), anti-INCENP antibody (rabbit, I5283, 1:2,000; Sigma-Aldrich), and anti-H3T3-Phos antibody (rabbit, ab78351, 1:1,000; Abcam).

    Techniques: Staining, Isolation, Western Blot, Control, Standard Deviation