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Tocris nocodazole treatment

(A) Schematic representation of human CENP-C. The Mis12C-binding domain (M12BD, amino acids 1–71) is highlighted in CENP-C WT. The N-terminal region (amino acids 1–75) was deleted in CENP-C ∆M12BD . FLAG-tagged CENP-C WT or ∆M12BD was introduced into the CENP-C locus in RPE-1 cells expressing mScarlet-CENP-A and GFP-H2A (CENP-C WT or CENP-C ∆M12BD RPE-1 cells, respectively; see ). (B) DSN1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (C) KNL1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. KNL1 was stained with an antibody against KNL1. KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD cells: n = 10. (D) Hec1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Hec1 was stained with an antibody against Hec1. Hec1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10. (E) Representative time-lapse images of mitotic progression in CENP-C WT or CENP-C ∆M12BD cells. DNA was visualized with GFP-H2A. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (F) Mitotic duration from the NEBD to the anaphase onset in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset. Two independent clones of CENP-C WT or CENP-C ∆M12BD RPE-1 cells were tested (mean and SD, two-tailed t test, CENP-C WT RPE-1 cell clone1: n = 120; CENP-C WT RPE-1 cell clone2: n = 105; CENP-C ∆M12BD RPE-1 cell clone1: n = 105; CENP-C ∆M12BD RPE-1 cell clone2: n = 132). (G) Chromosome segregation errors in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (F) were scored. Representative images are shown. Scale bar, 5 μm. (H) Micronucleus formation in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The cells were fixed, and the interphase cells with micronuclei were scored (CENP-C WT RPE-1 cell clone1: n = 1,527; CENP-C WT RPE-1 cell clone2: n = 976; CENP-C ∆M12BD RPE-1 cell clone1: n = 1,633; CENP-C ∆M12BD RPE-1 cell clone2: n = 1,076). Scale bar, 10 μm. (I) K-fiber in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells expressing mScarlet CENP-A were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody. Scale bar, 5 μm. The means of tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (J) Cell viability of CENP-C WT or CENP-C ∆M12BD RPE-1 cells treated with various concentrations of <t>nocodazole.</t> Viable cells were measured 3 d after nocodazole addition. Three independent experiments were performed (mean and SD, two-way ANOVA with Šídák’s multiple comparison test, ** P = 0.0022). IC 50 indicates the average of concentration to reduce cell viability to 50% from three independent experiments (mean and SD, two-tailed t test).

Nocodazole Treatment, supplied by Tocris, used in various techniques. Bioz Stars score: 95/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation"

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

Journal: Life Science Alliance

doi: 10.26508/lsa.202402927

Figure Legend Snippet: (A) Schematic representation of human CENP-C. The Mis12C-binding domain (M12BD, amino acids 1–71) is highlighted in CENP-C WT. The N-terminal region (amino acids 1–75) was deleted in CENP-C ∆M12BD . FLAG-tagged CENP-C WT or ∆M12BD was introduced into the CENP-C locus in RPE-1 cells expressing mScarlet-CENP-A and GFP-H2A (CENP-C WT or CENP-C ∆M12BD RPE-1 cells, respectively; see ). (B) DSN1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (C) KNL1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. KNL1 was stained with an antibody against KNL1. KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD cells: n = 10. (D) Hec1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Hec1 was stained with an antibody against Hec1. Hec1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10. (E) Representative time-lapse images of mitotic progression in CENP-C WT or CENP-C ∆M12BD cells. DNA was visualized with GFP-H2A. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (F) Mitotic duration from the NEBD to the anaphase onset in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset. Two independent clones of CENP-C WT or CENP-C ∆M12BD RPE-1 cells were tested (mean and SD, two-tailed t test, CENP-C WT RPE-1 cell clone1: n = 120; CENP-C WT RPE-1 cell clone2: n = 105; CENP-C ∆M12BD RPE-1 cell clone1: n = 105; CENP-C ∆M12BD RPE-1 cell clone2: n = 132). (G) Chromosome segregation errors in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (F) were scored. Representative images are shown. Scale bar, 5 μm. (H) Micronucleus formation in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The cells were fixed, and the interphase cells with micronuclei were scored (CENP-C WT RPE-1 cell clone1: n = 1,527; CENP-C WT RPE-1 cell clone2: n = 976; CENP-C ∆M12BD RPE-1 cell clone1: n = 1,633; CENP-C ∆M12BD RPE-1 cell clone2: n = 1,076). Scale bar, 10 μm. (I) K-fiber in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells expressing mScarlet CENP-A were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody. Scale bar, 5 μm. The means of tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (J) Cell viability of CENP-C WT or CENP-C ∆M12BD RPE-1 cells treated with various concentrations of nocodazole. Viable cells were measured 3 d after nocodazole addition. Three independent experiments were performed (mean and SD, two-way ANOVA with Šídák’s multiple comparison test, ** P = 0.0022). IC 50 indicates the average of concentration to reduce cell viability to 50% from three independent experiments (mean and SD, two-tailed t test).

Techniques Used: Binding Assay, Expressing, Staining, Marker, Two Tailed Test, Clone Assay, Comparison, Concentration Assay

(A) Schematic representation of human CENP-T. Human CENP-T WT has two Ndc80C-binding regions (NBD-1 or NBD-2: amino acids 6–31 or 76–105) and a Mis12-binding domain. Each NBD was deleted in CENP-T ∆NBD−1 or CENP-T ∆NBD−2 RPE-1 cells, respectively. mScarlet-fused CENP-T WT or each mutant was introduced into the CENP-T locus in RPE-1 cells expressing OsTIR1 and GFP-mAID-CENP-T (CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells, respectively; see ). (B) Hec1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Hec1 was stained with an antibody against Hec1 (green). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Hec1 signal intensities at mitotic kinetochores were quantified (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (C) DSN1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). DSN1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 cells: n = 10. (D) KNL1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10. (E) K-fiber in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody (green). CENP-T fused with mScarlet was used as a kinetochore marker (CENP-T, red). Scale bar, 5 μm. The means of tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (F) Cell viability of CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells treated with various concentrations of nocodazole. Viable cells were measured 3 d after nocodazole addition. Four independent experiments were performed (mean and SD, two-way ANOVA with Dunnett’s multiple comparison test, *** P = 0.0007, **** P < 0.0001, red: WT versus ∆NBD-1; blue: WT versus ∆NBD-2). IC 50 indicates the average of nocodazole concentration to reduce cell viability to 50% from four independent experiments (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test). (G) Representative time-lapse images of mitotic progression in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DNA was visualized with SPY505-DNA. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (H) Mitotic duration from the NEBD to the anaphase onset in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 117; CENP-T ∆NBD−1 RPE-1 cells: n = 120; CENP-T ∆NBD−2 RPE-1 cells: n = 110). (I) Chromosome segregation errors in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (H) were scored. Representative images are shown. Scale bar, 5 μm.

Figure Legend Snippet: (A) Schematic representation of human CENP-T. Human CENP-T WT has two Ndc80C-binding regions (NBD-1 or NBD-2: amino acids 6–31 or 76–105) and a Mis12-binding domain. Each NBD was deleted in CENP-T ∆NBD−1 or CENP-T ∆NBD−2 RPE-1 cells, respectively. mScarlet-fused CENP-T WT or each mutant was introduced into the CENP-T locus in RPE-1 cells expressing OsTIR1 and GFP-mAID-CENP-T (CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells, respectively; see ). (B) Hec1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Hec1 was stained with an antibody against Hec1 (green). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Hec1 signal intensities at mitotic kinetochores were quantified (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (C) DSN1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). DSN1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 cells: n = 10. (D) KNL1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10. (E) K-fiber in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody (green). CENP-T fused with mScarlet was used as a kinetochore marker (CENP-T, red). Scale bar, 5 μm. The means of tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (F) Cell viability of CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells treated with various concentrations of nocodazole. Viable cells were measured 3 d after nocodazole addition. Four independent experiments were performed (mean and SD, two-way ANOVA with Dunnett’s multiple comparison test, *** P = 0.0007, **** P < 0.0001, red: WT versus ∆NBD-1; blue: WT versus ∆NBD-2). IC 50 indicates the average of nocodazole concentration to reduce cell viability to 50% from four independent experiments (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test). (G) Representative time-lapse images of mitotic progression in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DNA was visualized with SPY505-DNA. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (H) Mitotic duration from the NEBD to the anaphase onset in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 117; CENP-T ∆NBD−1 RPE-1 cells: n = 120; CENP-T ∆NBD−2 RPE-1 cells: n = 110). (I) Chromosome segregation errors in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (H) were scored. Representative images are shown. Scale bar, 5 μm.

Techniques Used: Binding Assay, Mutagenesis, Expressing, Staining, Marker, Comparison, Concentration Assay

(A) BubR1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. BubR1 was stained with an antibody against BubR1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. BubR1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 323 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 363 kinetochores from 5 cells). (B) Ska3 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Ska3 was stained with an antibody against Ska3 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Ska3 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 419 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 422 kinetochores from 5 cells). (C) PLK1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells were synchronized using a double thymidine block. After the washout of the second thymidine treatment for 6 h, the cells were treated with 1 μg/ml nocodazole for 4 h, as indicated in the scheme. PLK1 was stained with an antibody against PLK1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. The insets show an enlarged pair kinetochore (scale bar, 1 μm). PLK1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 359 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 361 kinetochores from 5 cells).

Figure Legend Snippet: (A) BubR1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. BubR1 was stained with an antibody against BubR1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. BubR1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 323 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 363 kinetochores from 5 cells). (B) Ska3 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Ska3 was stained with an antibody against Ska3 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Ska3 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 419 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 422 kinetochores from 5 cells). (C) PLK1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells were synchronized using a double thymidine block. After the washout of the second thymidine treatment for 6 h, the cells were treated with 1 μg/ml nocodazole for 4 h, as indicated in the scheme. PLK1 was stained with an antibody against PLK1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. The insets show an enlarged pair kinetochore (scale bar, 1 μm). PLK1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 359 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 361 kinetochores from 5 cells).

Techniques Used: Staining, Marker, Two Tailed Test, Blocking Assay

other:

Article Title: H3K27ac bookmarking promotes rapid post-mitotic activation of the pluripotent stem cell program without impacting 3D chromatin reorganization
Article Snippet: After 3 hours of Nocodazole treatment, either 20uM p300/CBP catalytic inhibitor (A485, Tocris #6387) or the same volume DMSO was added to the media for the remaining 3 hours of synchronization.

Article Title: Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression.
Article Snippet: The sgRNA of TP53 (5’- CCGGTTCATGCCGCCCATGC-3’)75 was cloned in the lentiCRISPR v2 vector (addgene 52961)75 to construct lentiCRISPR-TP53 for making lentiviruses.

Modification:

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation
Article Snippet: The cells were cultured with 100 ng/ml nocodazole (Sigma-Aldrich) for 2–4 h at 37°C. .. To detect Aurora B at kinetochore-proximal pool , nocodazole treatment followed with 5 μM 5-iodotubercidin (5-ITu; TOCRIS), a Haspin inhibitor, for 30 min. To spread and swell chromosomes, the method was modified from the protocol described previously ( Preprint ). .. After shaking off and collecting the mitotic-arrested cells, they were suspended in a hypotonic buffer (75 mM KCl [Nacalai Tesque]: 0.8% sodium citrate [Nacalai Tesque]: water at 1:1:1) with 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) on ice.

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation.
Article Snippet: The cells were cultured with 100 ng/ ml nocodazole (Sigma-Aldrich) for 2–4 h at 37°C. .. To detect Aurora B at kinetochore-proximal pool (Fig S8H), nocodazole treatment followed with 5 μM 5-iodotubercidin (5-ITu; TOCRIS), a Haspin inhibitor, for 30 min. To spread and swell chromosomes, the method was modified from the protocol described previously (Erin et al, 2021 Preprint). .. After shaking off and collecting the mitotic-arrested cells, they were suspended in a hypotonic buffer (75 mM KCl [Nacalai Tesque]: 0.8% sodium citrate [Nacalai Tesque]: water at 1:1:1) with 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) on ice.

Image Search Results

S277 is a CDK2 target in cells . A , schematic of the RAD51AP1 protein (isoform 2) with predicted CDK1/2 sites (GPS 6.0). The F3 fragment (residues 188–335) and the bipartite DNA binding domain ( gray ) are indicated. Predicted CDK1/2 sites in F3 (S224, S277 and S310) were changed to alanine. B , results from kinase reaction of purified His 6 -F3-FLAG and mutants (S224/310A and S224/277/310A) with CDK2/Cyclin E2 and ɣ- 32 P-ATP. Upper : Phosphorimage; lower : Western blot with ⍺-FLAG antibody. C , results from kinase reaction of purified His 6 -F3-FLAG and mutants (S224/310A and S224/277/310A) with CDK1/Cyclin B1 and ɣ- 32 P-ATP. Upper : Phosphorimage; lower : Western blot with ⍺-FLAG antibody. D , results from kinase reaction of stably expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-RAD51AP1 and the FLAG-RAD51AP1-S2A (S277/282A) mutant with CDK2/Cyclin E2 or CDK1/Cyclin B1 and ɣ- 32 P-ATP. Upper : Phosphorimage; middle : Phosphorimage; lower : Western blot with ⍺-RAD51AP1 antibody. The product of the kinase reaction with purified (from E . coli ) His6-RAD51AP1-FLAG protein is shown for comparison purposes (rRAD51AP1; lane 1). E , Results from kinase reaction of transiently expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-F3 and mutants (S224/310A and S224/277/310A) with CDK2/Cyclin E2 and ɣ- 32 P-ATP. Upper : Phosphorimage; middle and lower : Western blot with ⍺-FLAG antibody. The product of the kinase reaction with purified (from E . coli ) His 6 -F3-FLAG is shown for comparison purposes (rF3; lane 1). Pink boxes : to highlight phosphorylated F3. F , results from kinase reaction of transiently expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-F3 with CDK2/Cyclin E2 and ɣ- 32 P-ATP without and with the addition of 1 μM Roscovitine (+R; lanes 6 and 7, respectively). The product of the kinase reaction with purified (from E . coli ) His 6 -F3-FLAG is shown for comparison purposes (rF3; lane 1). G , CDK2 and Cyclin A are present in ⍺-FLAG precipitates of FLAG-RAD51AP1 (WT) stably expressed in HeLa RAD51AP1 KO cells, synchronized by a double thymidine block (DTB) and released into early S phase (lane 3). CDK2 and Cyclin A are not present FLAG-RAD51AP1 (WT) precipitates from cells arrested in late G2/M phase by treatment with nocodazole (NCO; lane 4). Note: In G2/M phase cells (lane 4), a partial mobility shift is observed for RAD51AP1 that—based on our preliminary results—is unrelated to S277/S282 phosphorylation (data not shown). H , CDK1 is not present in FLAG-RAD51AP1 (WT) precipitates of cells synchronized in early S (lane 3) or late G2/M phase (lane 4). I , CDK2 and Cyclin A are present in precipitates of transiently expressed FLAG-F3 and mutants in RAD51AP1 KO cells synchronized by a DTB and released into early S phase (lanes 2–4).

Journal: The Journal of Biological Chemistry

Article Title: Phosphoregulation of RAD51AP1 function in homology-directed repair

doi: 10.1016/j.jbc.2026.111149

Figure Lengend Snippet: S277 is a CDK2 target in cells . A , schematic of the RAD51AP1 protein (isoform 2) with predicted CDK1/2 sites (GPS 6.0). The F3 fragment (residues 188–335) and the bipartite DNA binding domain ( gray ) are indicated. Predicted CDK1/2 sites in F3 (S224, S277 and S310) were changed to alanine. B , results from kinase reaction of purified His 6 -F3-FLAG and mutants (S224/310A and S224/277/310A) with CDK2/Cyclin E2 and ɣ- 32 P-ATP. Upper : Phosphorimage; lower : Western blot with ⍺-FLAG antibody. C , results from kinase reaction of purified His 6 -F3-FLAG and mutants (S224/310A and S224/277/310A) with CDK1/Cyclin B1 and ɣ- 32 P-ATP. Upper : Phosphorimage; lower : Western blot with ⍺-FLAG antibody. D , results from kinase reaction of stably expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-RAD51AP1 and the FLAG-RAD51AP1-S2A (S277/282A) mutant with CDK2/Cyclin E2 or CDK1/Cyclin B1 and ɣ- 32 P-ATP. Upper : Phosphorimage; middle : Phosphorimage; lower : Western blot with ⍺-RAD51AP1 antibody. The product of the kinase reaction with purified (from E . coli ) His6-RAD51AP1-FLAG protein is shown for comparison purposes (rRAD51AP1; lane 1). E , Results from kinase reaction of transiently expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-F3 and mutants (S224/310A and S224/277/310A) with CDK2/Cyclin E2 and ɣ- 32 P-ATP. Upper : Phosphorimage; middle and lower : Western blot with ⍺-FLAG antibody. The product of the kinase reaction with purified (from E . coli ) His 6 -F3-FLAG is shown for comparison purposes (rF3; lane 1). Pink boxes : to highlight phosphorylated F3. F , results from kinase reaction of transiently expressed (in HeLa RAD51AP1 KO cells) and precipitated FLAG-F3 with CDK2/Cyclin E2 and ɣ- 32 P-ATP without and with the addition of 1 μM Roscovitine (+R; lanes 6 and 7, respectively). The product of the kinase reaction with purified (from E . coli ) His 6 -F3-FLAG is shown for comparison purposes (rF3; lane 1). G , CDK2 and Cyclin A are present in ⍺-FLAG precipitates of FLAG-RAD51AP1 (WT) stably expressed in HeLa RAD51AP1 KO cells, synchronized by a double thymidine block (DTB) and released into early S phase (lane 3). CDK2 and Cyclin A are not present FLAG-RAD51AP1 (WT) precipitates from cells arrested in late G2/M phase by treatment with nocodazole (NCO; lane 4). Note: In G2/M phase cells (lane 4), a partial mobility shift is observed for RAD51AP1 that—based on our preliminary results—is unrelated to S277/S282 phosphorylation (data not shown). H , CDK1 is not present in FLAG-RAD51AP1 (WT) precipitates of cells synchronized in early S (lane 3) or late G2/M phase (lane 4). I , CDK2 and Cyclin A are present in precipitates of transiently expressed FLAG-F3 and mutants in RAD51AP1 KO cells synchronized by a DTB and released into early S phase (lanes 2–4).

Article Snippet: For treatment with Nocodazole (NCO; AdipoGen Life Sciences), cells were released into regular growth medium for 4 h after the DTB before NCO (80 ng/ml) was added, and cells were incubated for another 14 h before cell harvest and lysis.

Techniques: Binding Assay, Purification, Western Blot, Stable Transfection, Mutagenesis, Comparison, Blocking Assay, Mobility Shift, Phospho-proteomics

(A) Immunofluorescent staining of metaphase chromosome spreads. Mitotic HeLa cells were enriched by treatment with colcemid (100 ng/mL) for 18 h, followed by a 5-minute release, and then subjected to co-immunostaining using anti-BubR1 and anti-acetylated BubR1 (AcK250) monoclonal antibodies (mAbs). Kinetochores were categorized into three groups based on anti-AcK250 mAb immunostaining: those immunostained at both sister kinetochores (Paired); one kinetochore (Single); or neither (No). Scale bar, 5 μm. (B) Proportion of anti-AcK250 mAb immunostaining patterns in (A). Results are from two independent experiments (mean ± s.e.m.; n = 40 chromosomes). P values were obtained using a t-test. (C) Representative images of microtubule attachment at kinetochores using structured illumination microscopy (SIM). Mitotic HeLa cells were treated with 10 μM MG132 to enrich the metaphase population, fixed, and then co-immunostained using anti-α-tubulin, anti-AcK250, and CREST antibodies. CREST-marked kinetochores were divided into three groups based on 3D-reconstructed images: unattached kinetochore (No attachment), monotelic kinetochore (Single-attachment), and amphitelic kinetochore (Bi-attachment). Scale bar, 5 μm (Top). Bottom, 3D-reconstructed images of anti-AcK250 mAb staining. Scale bar, 0.5 μm. The SIM images were acquired using an AP DeltaVision OMX Ultra High-Resolution Fluorescence Microscope (GE Healthcare). Image reconstruction and alignment were performed using SoftWoRx software. Three-dimensional reconstruction was performed using ImageJ software (3D projection). (D) Relative intensity of anti-AcK250 immunostaining normalized to CREST. Number of kinetochores scored: No attachment (No), n = 20; monotelic attachment (Single), n = 22; bipolar attachment (Bi), n = 16. The graphs represent two independent experiments (mean ± s.e.m.). Statistical differences between groups were assessed using the Mann-Whitney U test; p values are indicated. (E-H) HeLa cells treated with 200 ng/mL nocodazole (Noc), 2 μM paclitaxel/taxol (Tax), 100 μM Monastrol (Mon), or 10 μM MG132, respectively, were co-immunostained with CREST, anti-α-tubulin, and anti-AcK250 (E, F) or anti-BubR1 (G, H). (F, H) Graphs are from two independent experiments (mean±s.e.m.). Scale bar, 5 μm.

Journal: bioRxiv

Article Title: Linking Kinetochore Attachment to Checkpoint Control: The Role of Aurora B in BubR1 Acetylation

doi: 10.1101/2025.08.22.671725

Figure Lengend Snippet: (A) Immunofluorescent staining of metaphase chromosome spreads. Mitotic HeLa cells were enriched by treatment with colcemid (100 ng/mL) for 18 h, followed by a 5-minute release, and then subjected to co-immunostaining using anti-BubR1 and anti-acetylated BubR1 (AcK250) monoclonal antibodies (mAbs). Kinetochores were categorized into three groups based on anti-AcK250 mAb immunostaining: those immunostained at both sister kinetochores (Paired); one kinetochore (Single); or neither (No). Scale bar, 5 μm. (B) Proportion of anti-AcK250 mAb immunostaining patterns in (A). Results are from two independent experiments (mean ± s.e.m.; n = 40 chromosomes). P values were obtained using a t-test. (C) Representative images of microtubule attachment at kinetochores using structured illumination microscopy (SIM). Mitotic HeLa cells were treated with 10 μM MG132 to enrich the metaphase population, fixed, and then co-immunostained using anti-α-tubulin, anti-AcK250, and CREST antibodies. CREST-marked kinetochores were divided into three groups based on 3D-reconstructed images: unattached kinetochore (No attachment), monotelic kinetochore (Single-attachment), and amphitelic kinetochore (Bi-attachment). Scale bar, 5 μm (Top). Bottom, 3D-reconstructed images of anti-AcK250 mAb staining. Scale bar, 0.5 μm. The SIM images were acquired using an AP DeltaVision OMX Ultra High-Resolution Fluorescence Microscope (GE Healthcare). Image reconstruction and alignment were performed using SoftWoRx software. Three-dimensional reconstruction was performed using ImageJ software (3D projection). (D) Relative intensity of anti-AcK250 immunostaining normalized to CREST. Number of kinetochores scored: No attachment (No), n = 20; monotelic attachment (Single), n = 22; bipolar attachment (Bi), n = 16. The graphs represent two independent experiments (mean ± s.e.m.). Statistical differences between groups were assessed using the Mann-Whitney U test; p values are indicated. (E-H) HeLa cells treated with 200 ng/mL nocodazole (Noc), 2 μM paclitaxel/taxol (Tax), 100 μM Monastrol (Mon), or 10 μM MG132, respectively, were co-immunostained with CREST, anti-α-tubulin, and anti-AcK250 (E, F) or anti-BubR1 (G, H). (F, H) Graphs are from two independent experiments (mean±s.e.m.). Scale bar, 5 μm.

Article Snippet: For kinase inhibition assays, the following inhibitors were added during the final 2 hours of a 20-hour nocodazole treatment: BI2536 (10nM, S1109; Selleckchem), Reversine (2μM, R3904; Sigma), Hesperadin (200nM, S1529; Selleckchem), and ZM447439 (2μM, S110301; Selleckchem).

Techniques: Staining, Immunostaining, Bioprocessing, Microscopy, Fluorescence, Software, MANN-WHITNEY

(A) Representative images of M-phase HeLa cells immunostained with anti-BubR1, CREST, and α-tubulin antibodies. Cells were treated with 10 μM MG132 to enrich the metaphase population. Kinetochores were categorized into three groups based on 3D-reconstructed SIM images: No attachment, Single-attachment, or Bi-attachment. Scale bar, 5 μm. Bottom, 3D reconstruction. Scale bar, 0.5 μm. (B) Volume of BubR1 in cells exhibiting no attachment (No), monotelic attachment (Single), and amphitelic attachment (Bi). Number of chromosomes scored: No, n = 131; Single, n = 89; Bi, n = 115. Results are from three independent experiments (mean ± s.e.m.). (C) Representative SIM images of FLAG-tagged BubR1-WT, -K250R, and -K250Q in mitotic kinetochores. HeLa-FRT-TO cells expressing a single copy of FLAG-BubR1, -K250R, or -K250Q were depleted of endogenous BubR1 via siRNA transfection. Simultaneously, doxycycline was used to induce the expression of BubR1-WT, -K250R, and -K250Q, respectively. Cells were treated with nocodazole (Noc) alone or with MG132 (Noc + MG132) to assess the effect without interference from the stability of K250R. Cells were treated with nocodazole (200 ng/mL, 20 h) alone or in combination with MG132 (10 μM, final 2 h). Scale bar, 5 μm. (D) Volume of FLAG (Left) and relative intensities of FLAG to CENP-A (Right) in nocodazole-treated cells with or without MG132, as shown in the graphs. For nocodazole-only treatment: WT, n = 12; K250R, n = 14; K250Q, n = 14. For nocodazole + MG132 treatment: WT, n = 72, K250R, n = 72, K250Q, n = 72. The graph represents two independent experiments (mean ± s.e.m.).

Journal: bioRxiv

Article Title: Linking Kinetochore Attachment to Checkpoint Control: The Role of Aurora B in BubR1 Acetylation

doi: 10.1101/2025.08.22.671725

Figure Lengend Snippet: (A) Representative images of M-phase HeLa cells immunostained with anti-BubR1, CREST, and α-tubulin antibodies. Cells were treated with 10 μM MG132 to enrich the metaphase population. Kinetochores were categorized into three groups based on 3D-reconstructed SIM images: No attachment, Single-attachment, or Bi-attachment. Scale bar, 5 μm. Bottom, 3D reconstruction. Scale bar, 0.5 μm. (B) Volume of BubR1 in cells exhibiting no attachment (No), monotelic attachment (Single), and amphitelic attachment (Bi). Number of chromosomes scored: No, n = 131; Single, n = 89; Bi, n = 115. Results are from three independent experiments (mean ± s.e.m.). (C) Representative SIM images of FLAG-tagged BubR1-WT, -K250R, and -K250Q in mitotic kinetochores. HeLa-FRT-TO cells expressing a single copy of FLAG-BubR1, -K250R, or -K250Q were depleted of endogenous BubR1 via siRNA transfection. Simultaneously, doxycycline was used to induce the expression of BubR1-WT, -K250R, and -K250Q, respectively. Cells were treated with nocodazole (Noc) alone or with MG132 (Noc + MG132) to assess the effect without interference from the stability of K250R. Cells were treated with nocodazole (200 ng/mL, 20 h) alone or in combination with MG132 (10 μM, final 2 h). Scale bar, 5 μm. (D) Volume of FLAG (Left) and relative intensities of FLAG to CENP-A (Right) in nocodazole-treated cells with or without MG132, as shown in the graphs. For nocodazole-only treatment: WT, n = 12; K250R, n = 14; K250Q, n = 14. For nocodazole + MG132 treatment: WT, n = 72, K250R, n = 72, K250Q, n = 72. The graph represents two independent experiments (mean ± s.e.m.).

Techniques: Expressing, Transfection

(A) Representative images of immunostaining with anti-Mad2 and anti-ZW10 antibodies after induction of BubR1-WT, -K250R, and -K250Q expression in HeLa-FRT-TO cells. Nocodazole (200 ng/mL) alone or with MG132 was applied before fixation. Endogenous BubR1 was depleted via siRNA. Number of cells scored for Mad2: WT, n = 15; K250R, n = 20; K250Q, n = 18. For ZW10: WT, n = 12; K250R, n = 14; K250Q, n = 14 (mean ± s.e.m.). Scale bar, 5 μm. (B) Representative SIM images of immunostained ZW10 at kinetochores of HeLa cells expressing FLAG-tagged BubR1-WT, -K250R, or -K250Q following double thymidine block and release. FRT/TO HeLa cells were treated with doxycycline for 55 hours to induce expression of ectopic BubR1 variants. Endogenous BubR1 was depleted using siRNA targeting the 3’UTR. Cells were fixed for immunostaining 10 h after the final thymidine release to enrich the mitotic population. (C) Effects of BubR1 acetylation status on CENP-E and RZZ complex formation. Cells were treated with nocodazole alone (+Noc) or with MG132 (+Noc +MG132) to assess the binding affinity without interference from the stability of K250R. Relative protein intensities were measured using a densitometer and are shown in blue. (D) Representative images showing immunostained ZW10 and anti-CENP-A antibodies in FRT/TO HeLa cells expressing BubR1-WT, K250R, or K250Q with (siCtrl) or without CENP-E (siCENP-E). Cells were transfected with siRNA targeting luciferase (siCtrl) or CENP-E (siCENP-E) for 48 hours prior to fixation. Nocodazole was administered for 20 hours before analysis.

Journal: bioRxiv

Article Title: Linking Kinetochore Attachment to Checkpoint Control: The Role of Aurora B in BubR1 Acetylation

doi: 10.1101/2025.08.22.671725

Figure Lengend Snippet: (A) Representative images of immunostaining with anti-Mad2 and anti-ZW10 antibodies after induction of BubR1-WT, -K250R, and -K250Q expression in HeLa-FRT-TO cells. Nocodazole (200 ng/mL) alone or with MG132 was applied before fixation. Endogenous BubR1 was depleted via siRNA. Number of cells scored for Mad2: WT, n = 15; K250R, n = 20; K250Q, n = 18. For ZW10: WT, n = 12; K250R, n = 14; K250Q, n = 14 (mean ± s.e.m.). Scale bar, 5 μm. (B) Representative SIM images of immunostained ZW10 at kinetochores of HeLa cells expressing FLAG-tagged BubR1-WT, -K250R, or -K250Q following double thymidine block and release. FRT/TO HeLa cells were treated with doxycycline for 55 hours to induce expression of ectopic BubR1 variants. Endogenous BubR1 was depleted using siRNA targeting the 3’UTR. Cells were fixed for immunostaining 10 h after the final thymidine release to enrich the mitotic population. (C) Effects of BubR1 acetylation status on CENP-E and RZZ complex formation. Cells were treated with nocodazole alone (+Noc) or with MG132 (+Noc +MG132) to assess the binding affinity without interference from the stability of K250R. Relative protein intensities were measured using a densitometer and are shown in blue. (D) Representative images showing immunostained ZW10 and anti-CENP-A antibodies in FRT/TO HeLa cells expressing BubR1-WT, K250R, or K250Q with (siCtrl) or without CENP-E (siCENP-E). Cells were transfected with siRNA targeting luciferase (siCtrl) or CENP-E (siCENP-E) for 48 hours prior to fixation. Nocodazole was administered for 20 hours before analysis.

Techniques: Immunostaining, Expressing, Blocking Assay, Binding Assay, Transfection, Luciferase

A) Immunostaining with anti-AcK250 mAb after treatment of nocodazole-arrested cells with the indicated mitotic kinase inhibitors: 10 nM BI2536 (Plk1 inhibitor), 2 μM Reversine (Mps1 inhibitor), 200 nM Hesperadin (Aurora B inhibitor), and 2 μM ZM447439 (Aurora B inhibitor). The kinase inhibitors were applied during the final 2 hours of a 20-hour nocodazole treatment. Scale bar, 5 μm. (B) Relative intensities of AcK250 at kinetochores from (A): Nocodazole-arrested cells without kinase inhibitor addition (NT), and nocodazole + BI2536-treated (BI2536), Reversine-treated (Rev), Hesperadin-treated (Hes), or ZM447439-treated (ZM) cells (mean ± s.e.m.). Results are from two independent experiments. (C) Assessing the effect of Aurora B kinase activity on MCC maintenance. HeLa cells were transfected with Aurora B-WT or kinase-dead mutant K106R-expressing constructs. Endogenous Aurora B was depleted using siRNA targeting the 3’UTR. BI2536 (10 nM), Reversine (2 μM), Hesperadin (200 nM), or ZM447439 (2 μM) were added during the final 2 hours of a 20-hour nocodazole treatment (Left). Immunoprecipitation was performed using an anti-CDC27 (APC3) antibody, followed by WB with indicated antibodies. To assess MCC stability, MG132 was added with the indicated kinase inhibitors in nocodazole-arrested cells (Right). WB of the total cell lysate (5%) is shown for the loading control (Bottom). (D) Representative images of anti-AcK250 immunostaining in cells expressing wild-type Aurora B (AurkB-WT) or kinase-dead Aurora B mutant (AurkB-K106R). Scale bar, 5 μm. (E) Intensity of AcK250 measured from (D). Number of cells counted: WT, n = 11; AurkB-K106R, n = 7 (mean ± s.e.m.). (F) Effect of Aurora B kinase activity on the expansion of BubR1 (Left) and ZW10 (Right) at nocodazole-treated kinetochores. Scale bar, 5 μm. (G) Volume of BubR1 and ZW10 at the kinetochores from (F). Number of cells scored: BubR1 in WT, n = 105; BubR1 in K106R, n = 121; ZW10 in WT, n = 153; ZW10 in K106R, n = 85 (mean ± s.e.m.). (H) Interaction between BubR1, Aurora B, and phosphorylated Aurora B (AurkB-pT232) was assessed by IP with anti-BubR1 and WB with indicated antibodies. Mitotic HeLa cells were collected by mitotic shake-off after 20 h of nocodazole treatment (M). Interphase attached cells (A) were used as a control.

Journal: bioRxiv

Article Title: Linking Kinetochore Attachment to Checkpoint Control: The Role of Aurora B in BubR1 Acetylation

doi: 10.1101/2025.08.22.671725

Figure Lengend Snippet: A) Immunostaining with anti-AcK250 mAb after treatment of nocodazole-arrested cells with the indicated mitotic kinase inhibitors: 10 nM BI2536 (Plk1 inhibitor), 2 μM Reversine (Mps1 inhibitor), 200 nM Hesperadin (Aurora B inhibitor), and 2 μM ZM447439 (Aurora B inhibitor). The kinase inhibitors were applied during the final 2 hours of a 20-hour nocodazole treatment. Scale bar, 5 μm. (B) Relative intensities of AcK250 at kinetochores from (A): Nocodazole-arrested cells without kinase inhibitor addition (NT), and nocodazole + BI2536-treated (BI2536), Reversine-treated (Rev), Hesperadin-treated (Hes), or ZM447439-treated (ZM) cells (mean ± s.e.m.). Results are from two independent experiments. (C) Assessing the effect of Aurora B kinase activity on MCC maintenance. HeLa cells were transfected with Aurora B-WT or kinase-dead mutant K106R-expressing constructs. Endogenous Aurora B was depleted using siRNA targeting the 3’UTR. BI2536 (10 nM), Reversine (2 μM), Hesperadin (200 nM), or ZM447439 (2 μM) were added during the final 2 hours of a 20-hour nocodazole treatment (Left). Immunoprecipitation was performed using an anti-CDC27 (APC3) antibody, followed by WB with indicated antibodies. To assess MCC stability, MG132 was added with the indicated kinase inhibitors in nocodazole-arrested cells (Right). WB of the total cell lysate (5%) is shown for the loading control (Bottom). (D) Representative images of anti-AcK250 immunostaining in cells expressing wild-type Aurora B (AurkB-WT) or kinase-dead Aurora B mutant (AurkB-K106R). Scale bar, 5 μm. (E) Intensity of AcK250 measured from (D). Number of cells counted: WT, n = 11; AurkB-K106R, n = 7 (mean ± s.e.m.). (F) Effect of Aurora B kinase activity on the expansion of BubR1 (Left) and ZW10 (Right) at nocodazole-treated kinetochores. Scale bar, 5 μm. (G) Volume of BubR1 and ZW10 at the kinetochores from (F). Number of cells scored: BubR1 in WT, n = 105; BubR1 in K106R, n = 121; ZW10 in WT, n = 153; ZW10 in K106R, n = 85 (mean ± s.e.m.). (H) Interaction between BubR1, Aurora B, and phosphorylated Aurora B (AurkB-pT232) was assessed by IP with anti-BubR1 and WB with indicated antibodies. Mitotic HeLa cells were collected by mitotic shake-off after 20 h of nocodazole treatment (M). Interphase attached cells (A) were used as a control.

Techniques: Immunostaining, Activity Assay, Transfection, Mutagenesis, Expressing, Construct, Immunoprecipitation, Control

(A) Identification of Aurora B-mediated phosphorylation sites of BubR1 in response to nocodazole treatment. (Top) Schematic illustration of BubR1, indicating potential Aurora B-mediated phosphorylation sites (Ser16 & Ser39) and the K250 acetylation site. (Bottom) IP-WB analysis to identify phosphorylation sites that crosstalk with K250 acetylation. mCherry-tagged BubR1 expression constructs were mutagenized in vitro to substitute the phosphorylation sites with alanine, based on proteome analysis (Supplementary Fig. S5), and then transfected into HeLa cells. Cells were treated with nocodazole (200 ng/mL) for 20 h and mitotic cells were collected by shake-off. Attached cells (Attach) were used as a control. Mitotic cell lysates, along with control lysates from attached cells (WT, Attach), were subjected to immunoprecipitation with anti-mCherry antibody, followed by WB with anti-AcK250 mAb. The same blot was reprobed with anti-BubR1 and anti-mCherry antibodies for normalization. Total cell lysates were subjected to WB with anti-phospho-H3 and anti-cyclin B antibodies to assess whether the cells were in mitosis. WB with anti-mCherry and anti-β-actin antibodies in TCL were used as a loading control. Relative band intensities (AcK250/BubR1) were measured using a densitometer and are indicated. (B) Effect of Ser16 or Ser39 phosphorylation on K250 acetylation. Immunofluorescence assay in cells expressing the indicated mCherry-tagged BubR1 variants. Nocodazole-treated cells were formaldehyde-fixed and subjected to immunostaining with anti-AcK250, anti-ACA (CREST), and anti-mCherry antibodies. (C) Graph showing the intensities of anti-AcK250 immunofluorescence from (B). Anti-AcK250 immunofluorescence signals were normalized to anti-mCherry. The results are from two independent experiments (WT, n = 30; K250R, n = 20; K250Q, n = 24; S16A, n = 28; S39A, n = 29) (mean ± s.e.m.). (D) Identification of the Aurora B-binding region in BubR1. IP with 9E10 (anti-Myc) and WB with anti-AurkB (Aurora B kinase) were performed. All BubR1-expressing constructs were Myc-tagged. The same blot was reprobed with 9E10 for normalization. Two percent of TCL was subjected to WB with the indicated antibodies. (E) In vitro phosphorylation assay to confirm the phosphorylation of S16 and S39 by AurKB. Recombinant BubR1 (1-150 a.a.), purified from E. coli, was subjected to in vitro phosphorylation by Aurora B immunoprecipitated from nocodazole-arrested HeLa cells. Attached cells were used as a control (Attach). [γ-32P]-ATP was used in the assay to facilitate detection. WB with anti-MBP and anti-AurkB show the loading control.

Journal: bioRxiv

Article Title: Linking Kinetochore Attachment to Checkpoint Control: The Role of Aurora B in BubR1 Acetylation

doi: 10.1101/2025.08.22.671725

Figure Lengend Snippet: (A) Identification of Aurora B-mediated phosphorylation sites of BubR1 in response to nocodazole treatment. (Top) Schematic illustration of BubR1, indicating potential Aurora B-mediated phosphorylation sites (Ser16 & Ser39) and the K250 acetylation site. (Bottom) IP-WB analysis to identify phosphorylation sites that crosstalk with K250 acetylation. mCherry-tagged BubR1 expression constructs were mutagenized in vitro to substitute the phosphorylation sites with alanine, based on proteome analysis (Supplementary Fig. S5), and then transfected into HeLa cells. Cells were treated with nocodazole (200 ng/mL) for 20 h and mitotic cells were collected by shake-off. Attached cells (Attach) were used as a control. Mitotic cell lysates, along with control lysates from attached cells (WT, Attach), were subjected to immunoprecipitation with anti-mCherry antibody, followed by WB with anti-AcK250 mAb. The same blot was reprobed with anti-BubR1 and anti-mCherry antibodies for normalization. Total cell lysates were subjected to WB with anti-phospho-H3 and anti-cyclin B antibodies to assess whether the cells were in mitosis. WB with anti-mCherry and anti-β-actin antibodies in TCL were used as a loading control. Relative band intensities (AcK250/BubR1) were measured using a densitometer and are indicated. (B) Effect of Ser16 or Ser39 phosphorylation on K250 acetylation. Immunofluorescence assay in cells expressing the indicated mCherry-tagged BubR1 variants. Nocodazole-treated cells were formaldehyde-fixed and subjected to immunostaining with anti-AcK250, anti-ACA (CREST), and anti-mCherry antibodies. (C) Graph showing the intensities of anti-AcK250 immunofluorescence from (B). Anti-AcK250 immunofluorescence signals were normalized to anti-mCherry. The results are from two independent experiments (WT, n = 30; K250R, n = 20; K250Q, n = 24; S16A, n = 28; S39A, n = 29) (mean ± s.e.m.). (D) Identification of the Aurora B-binding region in BubR1. IP with 9E10 (anti-Myc) and WB with anti-AurkB (Aurora B kinase) were performed. All BubR1-expressing constructs were Myc-tagged. The same blot was reprobed with 9E10 for normalization. Two percent of TCL was subjected to WB with the indicated antibodies. (E) In vitro phosphorylation assay to confirm the phosphorylation of S16 and S39 by AurKB. Recombinant BubR1 (1-150 a.a.), purified from E. coli, was subjected to in vitro phosphorylation by Aurora B immunoprecipitated from nocodazole-arrested HeLa cells. Attached cells were used as a control (Attach). [γ-32P]-ATP was used in the assay to facilitate detection. WB with anti-MBP and anti-AurkB show the loading control.

Techniques: Phospho-proteomics, Expressing, Construct, In Vitro, Transfection, Control, Immunoprecipitation, Immunofluorescence, Immunostaining, Binding Assay, Recombinant, Purification

(A) IP-WB analysis to assess the effect of Ser16 and Ser39 phosphorylation on MCC maintenance after nocodazole treatment. The indicated mCherry-tagged BubR1 variants were transfected, and endogenous BubR1 was depleted by co-transfection of siRNA for BubR1 targeting the 3’UTR. Immunoprecipitation was performed with an anti-mCherry antibody, followed by WB with the indicated antibodies. Results for nocodazole-arrested HeLa cells treated with MG132 for 2 hours before harvest are shown at right (+Noc +MG132). WB of 2% TCL is shown at the bottom. (B, C) Effects of phosphorylation and acetylation on MCC maintenance. (B) Effects of AurKB-mediated phosphorylation at Serines 16 and 39 on MCC maintenance. Phospho-deficient mutants S16A-BubR1 and S39A-BubR1, as well as the S16A;S39A mutant, and phospho-deficient mutations introduced into the acetylation-mimetic form (S16A;K250Q, S39A;K250Q, S16A;S39A;K250Q) were transfected into HeLa cells, along with siBubR1 to deplete endogenous BubR1. The cells were treated with nocodazole and subjected to IP with anti-mCherry antibody, followed by WB with the indicated antibodies. (C) Effect of the phospho-mimetic S16D-BubR1 or S39D-BubR1 on MCC maintenance. The acetylation-deficient K250R mutation was introduced into S16D or S39D (S16D;K250R and S39D;K250R) variants, and their capabilities for maintaining MCC after nocodazole treatment were assessed. All BubR1 variants were tagged with mCherry, transfected into HeLa cells, and subjected to IP and WB using the indicated antibodies. (D) Effect of AurKB-mediated phosphorylation and K250 acetylation on mitotic timing. Endogenous BubR1 was depleted using siRNA, and the indicated mCherry-tagged BubR1 variants were transfected into HeLa cells stably expressing H2B-GFP . Images were captured at 5-minute intervals. Arrows mark anaphase onset.

Journal: bioRxiv

Article Title: Linking Kinetochore Attachment to Checkpoint Control: The Role of Aurora B in BubR1 Acetylation

doi: 10.1101/2025.08.22.671725

Figure Lengend Snippet: (A) IP-WB analysis to assess the effect of Ser16 and Ser39 phosphorylation on MCC maintenance after nocodazole treatment. The indicated mCherry-tagged BubR1 variants were transfected, and endogenous BubR1 was depleted by co-transfection of siRNA for BubR1 targeting the 3’UTR. Immunoprecipitation was performed with an anti-mCherry antibody, followed by WB with the indicated antibodies. Results for nocodazole-arrested HeLa cells treated with MG132 for 2 hours before harvest are shown at right (+Noc +MG132). WB of 2% TCL is shown at the bottom. (B, C) Effects of phosphorylation and acetylation on MCC maintenance. (B) Effects of AurKB-mediated phosphorylation at Serines 16 and 39 on MCC maintenance. Phospho-deficient mutants S16A-BubR1 and S39A-BubR1, as well as the S16A;S39A mutant, and phospho-deficient mutations introduced into the acetylation-mimetic form (S16A;K250Q, S39A;K250Q, S16A;S39A;K250Q) were transfected into HeLa cells, along with siBubR1 to deplete endogenous BubR1. The cells were treated with nocodazole and subjected to IP with anti-mCherry antibody, followed by WB with the indicated antibodies. (C) Effect of the phospho-mimetic S16D-BubR1 or S39D-BubR1 on MCC maintenance. The acetylation-deficient K250R mutation was introduced into S16D or S39D (S16D;K250R and S39D;K250R) variants, and their capabilities for maintaining MCC after nocodazole treatment were assessed. All BubR1 variants were tagged with mCherry, transfected into HeLa cells, and subjected to IP and WB using the indicated antibodies. (D) Effect of AurKB-mediated phosphorylation and K250 acetylation on mitotic timing. Endogenous BubR1 was depleted using siRNA, and the indicated mCherry-tagged BubR1 variants were transfected into HeLa cells stably expressing H2B-GFP . Images were captured at 5-minute intervals. Arrows mark anaphase onset.

Techniques: Phospho-proteomics, Transfection, Cotransfection, Immunoprecipitation, Mutagenesis, Stable Transfection, Expressing

(A, B) Effects of AurkB-mediated phosphorylation and subsequent K250 acetylation on fibrous corona expansion. (A) SIM images of anti-Bub1 or anti-MAD2 immunostaining in nocodazole-treated cells are shown. The indicated BubR1 constructs were transfected with siBubR1 targeting the 3’UTR 48 hours before fixation, and nocodazole (200 ng/mL) was applied for 20 hours. Volumes were measured from the immunofluorescence assays shown. Number of cells scored: Bub1 in WT, n = 63; Bub1 in S16A, n = 64; Bub1 in S39A, n = 70; MAD2 in WT, n = 196; MAD2 in S16A, n = 196; MAD2 in S39A, n = 69 (mean ± s.e.m.). (B) SIM images of anti-ZW10 immunostaining in nocodazole-treated cells. Endogenous BubR1 was depleted via co-transfection with siBubR1. Nocodazole (200 ng/mL) was administered for 20 hours after 48 h of transfection. Insets show enlarged images of ZW10 and CREST staining. Scale bar, 5 μm. (C) Volume of ZW10 measured from the immunofluorescence assay shown in (B). Number of cells scored: WT, n = 27; S16A, n = 29; S39A, n = 27; S16A;K250Q, n = 31; S39A;K250Q, n = 32; K250Q, n = 29; S16D, n = 31; S39D, n = 32; S16D;K250R, n = 27; S39D;K250R, n = 27; K250R, n = 27 (mean ± s.e.m.). (D) Model illustrating the coordination of AurKB-mediated phosphorylation and K250 acetylation in spindle checkpoint signaling. At unattached kinetochores, AurKB phosphorylates BubR1 at S16 and S39, which promotes BubR1 acetylation at K250 by PCAF. This acetylation ensures the maintenance of the RZZ complex and MCC. It also sustains CENP-E at the kinetochore, preparing for switch from lateral to end-on attachment (Left). Upon successful end-on capture, PP2A activity recruited by AckK250-BubR1 antagonizes AurkB activity, stabilizing KT-MT attachment. Simultaneously, BubR1 immediately deacetylates and degraded through APC/C-mediated ubiquitination, resulting MCC disassembly and SAC silencing.

Journal: bioRxiv

Article Title: Linking Kinetochore Attachment to Checkpoint Control: The Role of Aurora B in BubR1 Acetylation

doi: 10.1101/2025.08.22.671725

Figure Lengend Snippet: (A, B) Effects of AurkB-mediated phosphorylation and subsequent K250 acetylation on fibrous corona expansion. (A) SIM images of anti-Bub1 or anti-MAD2 immunostaining in nocodazole-treated cells are shown. The indicated BubR1 constructs were transfected with siBubR1 targeting the 3’UTR 48 hours before fixation, and nocodazole (200 ng/mL) was applied for 20 hours. Volumes were measured from the immunofluorescence assays shown. Number of cells scored: Bub1 in WT, n = 63; Bub1 in S16A, n = 64; Bub1 in S39A, n = 70; MAD2 in WT, n = 196; MAD2 in S16A, n = 196; MAD2 in S39A, n = 69 (mean ± s.e.m.). (B) SIM images of anti-ZW10 immunostaining in nocodazole-treated cells. Endogenous BubR1 was depleted via co-transfection with siBubR1. Nocodazole (200 ng/mL) was administered for 20 hours after 48 h of transfection. Insets show enlarged images of ZW10 and CREST staining. Scale bar, 5 μm. (C) Volume of ZW10 measured from the immunofluorescence assay shown in (B). Number of cells scored: WT, n = 27; S16A, n = 29; S39A, n = 27; S16A;K250Q, n = 31; S39A;K250Q, n = 32; K250Q, n = 29; S16D, n = 31; S39D, n = 32; S16D;K250R, n = 27; S39D;K250R, n = 27; K250R, n = 27 (mean ± s.e.m.). (D) Model illustrating the coordination of AurKB-mediated phosphorylation and K250 acetylation in spindle checkpoint signaling. At unattached kinetochores, AurKB phosphorylates BubR1 at S16 and S39, which promotes BubR1 acetylation at K250 by PCAF. This acetylation ensures the maintenance of the RZZ complex and MCC. It also sustains CENP-E at the kinetochore, preparing for switch from lateral to end-on attachment (Left). Upon successful end-on capture, PP2A activity recruited by AckK250-BubR1 antagonizes AurkB activity, stabilizing KT-MT attachment. Simultaneously, BubR1 immediately deacetylates and degraded through APC/C-mediated ubiquitination, resulting MCC disassembly and SAC silencing.

Techniques: Phospho-proteomics, Immunostaining, Construct, Transfection, Immunofluorescence, Cotransfection, Staining, Activity Assay, Ubiquitin Proteomics

Journal: Life Science Alliance

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

doi: 10.26508/lsa.202402927

Figure Lengend Snippet: (A) Schematic representation of human CENP-C. The Mis12C-binding domain (M12BD, amino acids 1–71) is highlighted in CENP-C WT. The N-terminal region (amino acids 1–75) was deleted in CENP-C ∆M12BD . FLAG-tagged CENP-C WT or ∆M12BD was introduced into the CENP-C locus in RPE-1 cells expressing mScarlet-CENP-A and GFP-H2A (CENP-C WT or CENP-C ∆M12BD RPE-1 cells, respectively; see ). (B) DSN1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (C) KNL1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. KNL1 was stained with an antibody against KNL1. KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD cells: n = 10. (D) Hec1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Hec1 was stained with an antibody against Hec1. Hec1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10. (E) Representative time-lapse images of mitotic progression in CENP-C WT or CENP-C ∆M12BD cells. DNA was visualized with GFP-H2A. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (F) Mitotic duration from the NEBD to the anaphase onset in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset. Two independent clones of CENP-C WT or CENP-C ∆M12BD RPE-1 cells were tested (mean and SD, two-tailed t test, CENP-C WT RPE-1 cell clone1: n = 120; CENP-C WT RPE-1 cell clone2: n = 105; CENP-C ∆M12BD RPE-1 cell clone1: n = 105; CENP-C ∆M12BD RPE-1 cell clone2: n = 132). (G) Chromosome segregation errors in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (F) were scored. Representative images are shown. Scale bar, 5 μm. (H) Micronucleus formation in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The cells were fixed, and the interphase cells with micronuclei were scored (CENP-C WT RPE-1 cell clone1: n = 1,527; CENP-C WT RPE-1 cell clone2: n = 976; CENP-C ∆M12BD RPE-1 cell clone1: n = 1,633; CENP-C ∆M12BD RPE-1 cell clone2: n = 1,076). Scale bar, 10 μm. (I) K-fiber in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells expressing mScarlet CENP-A were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody. Scale bar, 5 μm. The means of tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (J) Cell viability of CENP-C WT or CENP-C ∆M12BD RPE-1 cells treated with various concentrations of nocodazole. Viable cells were measured 3 d after nocodazole addition. Three independent experiments were performed (mean and SD, two-way ANOVA with Šídák’s multiple comparison test, ** P = 0.0022). IC 50 indicates the average of concentration to reduce cell viability to 50% from three independent experiments (mean and SD, two-tailed t test).

Article Snippet: To detect Aurora B at kinetochore-proximal pool , nocodazole treatment followed with 5 μM 5-iodotubercidin (5-ITu; TOCRIS), a Haspin inhibitor, for 30 min. To spread and swell chromosomes, the method was modified from the protocol described previously ( Preprint ).

Techniques: Binding Assay, Expressing, Staining, Marker, Two Tailed Test, Clone Assay, Comparison, Concentration Assay

Journal: Life Science Alliance

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

doi: 10.26508/lsa.202402927

Figure Lengend Snippet: (A) Schematic representation of human CENP-T. Human CENP-T WT has two Ndc80C-binding regions (NBD-1 or NBD-2: amino acids 6–31 or 76–105) and a Mis12-binding domain. Each NBD was deleted in CENP-T ∆NBD−1 or CENP-T ∆NBD−2 RPE-1 cells, respectively. mScarlet-fused CENP-T WT or each mutant was introduced into the CENP-T locus in RPE-1 cells expressing OsTIR1 and GFP-mAID-CENP-T (CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells, respectively; see ). (B) Hec1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Hec1 was stained with an antibody against Hec1 (green). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Hec1 signal intensities at mitotic kinetochores were quantified (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (C) DSN1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). DSN1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 cells: n = 10. (D) KNL1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10. (E) K-fiber in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody (green). CENP-T fused with mScarlet was used as a kinetochore marker (CENP-T, red). Scale bar, 5 μm. The means of tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (F) Cell viability of CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells treated with various concentrations of nocodazole. Viable cells were measured 3 d after nocodazole addition. Four independent experiments were performed (mean and SD, two-way ANOVA with Dunnett’s multiple comparison test, *** P = 0.0007, **** P < 0.0001, red: WT versus ∆NBD-1; blue: WT versus ∆NBD-2). IC 50 indicates the average of nocodazole concentration to reduce cell viability to 50% from four independent experiments (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test). (G) Representative time-lapse images of mitotic progression in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DNA was visualized with SPY505-DNA. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (H) Mitotic duration from the NEBD to the anaphase onset in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 117; CENP-T ∆NBD−1 RPE-1 cells: n = 120; CENP-T ∆NBD−2 RPE-1 cells: n = 110). (I) Chromosome segregation errors in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (H) were scored. Representative images are shown. Scale bar, 5 μm.

Techniques: Binding Assay, Mutagenesis, Expressing, Staining, Marker, Comparison, Concentration Assay

Journal: Life Science Alliance

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

doi: 10.26508/lsa.202402927

Figure Lengend Snippet: (A) BubR1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. BubR1 was stained with an antibody against BubR1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. BubR1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 323 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 363 kinetochores from 5 cells). (B) Ska3 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Ska3 was stained with an antibody against Ska3 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Ska3 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 419 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 422 kinetochores from 5 cells). (C) PLK1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells were synchronized using a double thymidine block. After the washout of the second thymidine treatment for 6 h, the cells were treated with 1 μg/ml nocodazole for 4 h, as indicated in the scheme. PLK1 was stained with an antibody against PLK1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. The insets show an enlarged pair kinetochore (scale bar, 1 μm). PLK1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 359 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 361 kinetochores from 5 cells).

Techniques: Staining, Marker, Two Tailed Test, Blocking Assay

(A) RBN-2397, paclitaxel, and their combination promote microtubule stability in OVCAR4 cells. Representative immunofluorescent images of α-tubulin in OVCAR4 cells after treatment with DMSO, RBN-2397 (500 nM), paclitaxel (0.4 nM), or their combination and exposure to cold treatment or nocodazole. The experiment was performed 3 times to ensure reproducibility. Scale bar = 12.5 μm. (B) Violin plots quantifying α-tubulin staining from experiments shown in (A) in OVCAR4 cells untreated (left panel) , cold (middle panel) , or nocodazole treated (right panel) . Violins marked with different letters are significantly different, Ordinary one-way ANOVA test; n=3.

Journal: bioRxiv

Article Title: RBN-2397, a PARP7 Inhibitor, Synergizes with Paclitaxel to Inhibit Proliferation and Migration of Ovarian Cancer Cells

doi: 10.1101/2024.08.20.608802

Figure Lengend Snippet: (A) RBN-2397, paclitaxel, and their combination promote microtubule stability in OVCAR4 cells. Representative immunofluorescent images of α-tubulin in OVCAR4 cells after treatment with DMSO, RBN-2397 (500 nM), paclitaxel (0.4 nM), or their combination and exposure to cold treatment or nocodazole. The experiment was performed 3 times to ensure reproducibility. Scale bar = 12.5 μm. (B) Violin plots quantifying α-tubulin staining from experiments shown in (A) in OVCAR4 cells untreated (left panel) , cold (middle panel) , or nocodazole treated (right panel) . Violins marked with different letters are significantly different, Ordinary one-way ANOVA test; n=3.

Article Snippet: For cold treatment, cells were placed on ice for 45 min, washed with warm medium three times and allowed to recover in normal growth medium at 37°C for 15 min. For nocodazole treatment, nocodazole (MedChemExpress, HY-13520) was added at 6 μM for 1 hr.

Techniques: Staining

Review

nocodazole treatment (Tocris)

Structured Review

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