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Medema labs human bub1
Human Bub1, supplied by Medema labs, 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/product/human+bub1/pm36861883-193-11-17?v=Medema+labs
Average 90 stars, based on 1 article reviews

human bub1 - by Bioz Stars, 2026-07

90/100 stars

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Fig. 6 BUB1 depletion disrupts spheroid growth and highlights prognostic signiﬁcance in MPM. A Representative image of hanging drop assay. BUB1 depletion impaired the spheroid formation capacity of MPM cells. Spheroids (n = 13, for each condition) were imaged using a stereo microscope. Scale bar: 200 μm. B The Box–Whisker plot of BUB1 mRNA expression levels in GSE2549, GSE42977, GSE51024, and GSE117668 datasets. C Representative IHC images depicting different immunoreactivity scores (IRS, ranging from 0 to 6) of BUB1 staining in MPM tissues. IRS was calculated by the multiplication of proportion of positive cells and the intensity of staining. Scale bar: 20 μm. D IHC- based analysis of BUB1 protein expression in human MPM tumor tissues (n = 10) compared to noncancerous normal samples (n = 10). MM: mesothelioma of other tissues. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05, **p < 0.01. E The Kaplan–Meier survival plots of BUB1 in the TCGA MPM and GSE2549 datasets compare high gene expression (orange) relative to the low/medium or low patients (blue). p values are shown on the plots.

Journal: Cell death & disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma.

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: Fig. 6 BUB1 depletion disrupts spheroid growth and highlights prognostic signiﬁcance in MPM. A Representative image of hanging drop assay. BUB1 depletion impaired the spheroid formation capacity of MPM cells. Spheroids (n = 13, for each condition) were imaged using a stereo microscope. Scale bar: 200 μm. B The Box–Whisker plot of BUB1 mRNA expression levels in GSE2549, GSE42977, GSE51024, and GSE117668 datasets. C Representative IHC images depicting different immunoreactivity scores (IRS, ranging from 0 to 6) of BUB1 staining in MPM tissues. IRS was calculated by the multiplication of proportion of positive cells and the intensity of staining. Scale bar: 20 μm. D IHC- based analysis of BUB1 protein expression in human MPM tumor tissues (n = 10) compared to noncancerous normal samples (n = 10). MM: mesothelioma of other tissues. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05, **p < 0.01. E The Kaplan–Meier survival plots of BUB1 in the TCGA MPM and GSE2549 datasets compare high gene expression (orange) relative to the low/medium or low patients (blue). p values are shown on the plots.

Article Snippet: To achieve stable ectopic expression, the human BUB1 coding sequence was extracted from the pBI-GFP-Bub1-wt backbone (Addgene plasmid #16624) using NotI (R3189S, NEB) and BamHI (R3136S, NEB) restriction enzymes, and subcloned into the multiple cloning site (MCS) of the lentiviral overexpression vector, pHIV-Zsgreen (Addgene plasmid #18121).

Techniques: Microscopy, Whisker Assay, Expressing, Staining, Two Tailed Test, Gene Expression

Fig. 7 BUB1 overexpression ampliﬁes proliferative and malignant phenotypes. A BUB1 protein levels in empty vector (Vector) and BUB1 overexpression vector (BUB1) transduced H2052, H2452, and H28 cells. β-actin was used as a loading control. B Representative images showing increased 2D colony formation capacity of H2052, H2452, and H28 cell lines upon BUB1 overexpression. Colony formation assay was performed in triplicates in 12-well cell culture plates for 10–14 days. High-resolution images of the plates were acquired by LI-COR Odyssey CLx Imaging System. C Crystal violet intensity data showing the relative difference in 2D colony forming capacity of Vector and BUB1- overexpressing cells. Image Studio software was used to measure signal intensities. Bar graphs are presented as the mean ± SD of three replicates. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05 and **p < 0.01. D Representative images of BrdU incorporation assay identifying increased cell proliferation index (BrdU positivity, red, 12 h incubation) in BUB1-overexpressing H2052, H2452, and H28 cells compared to Vector control cells. DAPI was used as the nuclear counterstain (Blue). Scale bar: 100 μm. E BrdU-positive cell percentages are presented as the mean ± SD, n = 6, n = 5 for H2452. Two-tailed Student’s t-test was used for statistical analysis. **p < 0.01 and ***p < 0.001, ns not signiﬁcant. F Representative images of soft agar colony formation assay. Scale bar: 100 µm. G Bar graphs showing the number of colonies with a diameter greater than 35 µm. Data are presented as mean ± SD, n = 5 for H2052, n = 4 for H2452. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05 and **p < 0.01. Representative images of transwell migration (H) and invasion (J) assays upon BUB1 overexpression with their relative controls. Scale bar: 100 μm. Migrated (I) and invaded (K) number of cells per ﬁeld. ImageJ software was used for manual cell counting. Data are presented as the mean ± SD, n = 6. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: Cell death & disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma.

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: Fig. 7 BUB1 overexpression ampliﬁes proliferative and malignant phenotypes. A BUB1 protein levels in empty vector (Vector) and BUB1 overexpression vector (BUB1) transduced H2052, H2452, and H28 cells. β-actin was used as a loading control. B Representative images showing increased 2D colony formation capacity of H2052, H2452, and H28 cell lines upon BUB1 overexpression. Colony formation assay was performed in triplicates in 12-well cell culture plates for 10–14 days. High-resolution images of the plates were acquired by LI-COR Odyssey CLx Imaging System. C Crystal violet intensity data showing the relative difference in 2D colony forming capacity of Vector and BUB1- overexpressing cells. Image Studio software was used to measure signal intensities. Bar graphs are presented as the mean ± SD of three replicates. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05 and **p < 0.01. D Representative images of BrdU incorporation assay identifying increased cell proliferation index (BrdU positivity, red, 12 h incubation) in BUB1-overexpressing H2052, H2452, and H28 cells compared to Vector control cells. DAPI was used as the nuclear counterstain (Blue). Scale bar: 100 μm. E BrdU-positive cell percentages are presented as the mean ± SD, n = 6, n = 5 for H2452. Two-tailed Student’s t-test was used for statistical analysis. **p < 0.01 and ***p < 0.001, ns not signiﬁcant. F Representative images of soft agar colony formation assay. Scale bar: 100 µm. G Bar graphs showing the number of colonies with a diameter greater than 35 µm. Data are presented as mean ± SD, n = 5 for H2052, n = 4 for H2452. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05 and **p < 0.01. Representative images of transwell migration (H) and invasion (J) assays upon BUB1 overexpression with their relative controls. Scale bar: 100 μm. Migrated (I) and invaded (K) number of cells per ﬁeld. ImageJ software was used for manual cell counting. Data are presented as the mean ± SD, n = 6. Two-tailed Student’s t-test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

Techniques: Over Expression, Plasmid Preparation, Control, Colony Assay, Cell Culture, Imaging, Software, Two Tailed Test, BrdU Incorporation Assay, Incubation, Soft Agar Assay, Migration, Cell Counting

Fig. 8 BUB1 is indispensable for maintaining SAC integrity and cytokinesis. A Heatmap depicting the expression levels of BUB1 local network genes (highlighted in Fig. S9A) in BUB1 knockout (BUB1 KO) H2052 and H2452 cells. B BUB1, Cyclin B, Cyclin A, CDC20 and p21 protein levels in BUB1 WT vs BUB1 KO cells. β-actin was used as a loading control. C Localization of MAD1, MAD2, and SGO1 in mitotic MPM cells demonstrated through immunoﬂuorescence staining. Cells were synchronized with a 12 h thymidine block followed by a 12 h release in a complete culture medium or 150 mM nocodazole for H28 cells. The images were captured as z-stacks using Apotome 3 (Zeiss) and subsequently processed into stacked projections. D Time-lapse live cell microscopy illustrating mitotic progression of BUB1 knockout H2452 cells compared to control. Cells were stained with Hoechst and monitored using confocal microscopy by acquiring z-stacks every 10 min for 20 h. Representative image sequences were aligned on the time axis and presented in the ﬁgures.

Journal: Cell death & disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma.

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: Fig. 8 BUB1 is indispensable for maintaining SAC integrity and cytokinesis. A Heatmap depicting the expression levels of BUB1 local network genes (highlighted in Fig. S9A) in BUB1 knockout (BUB1 KO) H2052 and H2452 cells. B BUB1, Cyclin B, Cyclin A, CDC20 and p21 protein levels in BUB1 WT vs BUB1 KO cells. β-actin was used as a loading control. C Localization of MAD1, MAD2, and SGO1 in mitotic MPM cells demonstrated through immunoﬂuorescence staining. Cells were synchronized with a 12 h thymidine block followed by a 12 h release in a complete culture medium or 150 mM nocodazole for H28 cells. The images were captured as z-stacks using Apotome 3 (Zeiss) and subsequently processed into stacked projections. D Time-lapse live cell microscopy illustrating mitotic progression of BUB1 knockout H2452 cells compared to control. Cells were stained with Hoechst and monitored using confocal microscopy by acquiring z-stacks every 10 min for 20 h. Representative image sequences were aligned on the time axis and presented in the ﬁgures.

Techniques: Expressing, Knock-Out, Control, Staining, Blocking Assay, Microscopy, Confocal Microscopy

(A) Aurora B localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Aurora B was stained with an antibody against Aurora B (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 2 μm). Aurora B signal intensities at inner centromeres were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 188 centromeres from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 166 centromeres from 5 cells). (B) Aurora B localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Aurora B and DNA were stained as in (A). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). Scale bar, 5 μm. The Aurora B signal intensities were quantified (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 197 centromeres from 5 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 198 centromeres from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 225 centromeres from 5 cells). (C) Chromosome oscillation in CENP-C WT , CENP-C ∆M12BD , CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. The deviation from the average position (DAP) was calculated from the time-lapse images . The graphs display the median and quantile with max and min (+ indicates mean) (two-tailed t test, CENP-C WT RPE-1 cells: n = 54 kinetochore pairs from 12 cells; CENP-C ∆M12BD RPE-1 cells: n = 42 kinetochore pairs from 7 cells; one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 17 kinetochore pairs from 3 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 15 kinetochore pairs from 3 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 16 kinetochore pairs from 4 cells). (D) H2AT120ph localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. H2AT120ph was stained with an antibody against H2AT120ph (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 1 μm). H2AT120ph signal intensities at kinetochore-proximal centromeres were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 398 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 373 kinetochores from 5 cells). (E) H3T3ph localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. H3T3ph was stained with an antibody against H3T3ph. H3T3ph localization at centromeres was examined and quantified as in (D). Scale bar, 10 μm. The graph displays the mean and SD (two-tailed t test, CENP-C WT : n = 170 centromeres from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 204 centromeres from 5 cells). (F) Bub1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Bub1 was stained with an antibody against Bub1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 318 kinetochores from 5 cells [prophase], n = 354 kinetochores from 5 cells [prometaphase], n = 440 kinetochores from 5 cells [metaphase]; CENP-C ∆M12BD RPE-1 cells: n = 280 kinetochores from 5 cells [prophase], n = 423 kinetochores from 5 cells [prometaphase], n = 429 kinetochores from 5 cells [metaphase]).

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) Aurora B localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Aurora B was stained with an antibody against Aurora B (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 2 μm). Aurora B signal intensities at inner centromeres were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 188 centromeres from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 166 centromeres from 5 cells). (B) Aurora B localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Aurora B and DNA were stained as in (A). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). Scale bar, 5 μm. The Aurora B signal intensities were quantified (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 197 centromeres from 5 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 198 centromeres from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 225 centromeres from 5 cells). (C) Chromosome oscillation in CENP-C WT , CENP-C ∆M12BD , CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. The deviation from the average position (DAP) was calculated from the time-lapse images . The graphs display the median and quantile with max and min (+ indicates mean) (two-tailed t test, CENP-C WT RPE-1 cells: n = 54 kinetochore pairs from 12 cells; CENP-C ∆M12BD RPE-1 cells: n = 42 kinetochore pairs from 7 cells; one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 17 kinetochore pairs from 3 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 15 kinetochore pairs from 3 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 16 kinetochore pairs from 4 cells). (D) H2AT120ph localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. H2AT120ph was stained with an antibody against H2AT120ph (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 1 μm). H2AT120ph signal intensities at kinetochore-proximal centromeres were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 398 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 373 kinetochores from 5 cells). (E) H3T3ph localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. H3T3ph was stained with an antibody against H3T3ph. H3T3ph localization at centromeres was examined and quantified as in (D). Scale bar, 10 μm. The graph displays the mean and SD (two-tailed t test, CENP-C WT : n = 170 centromeres from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 204 centromeres from 5 cells). (F) Bub1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Bub1 was stained with an antibody against Bub1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 318 kinetochores from 5 cells [prophase], n = 354 kinetochores from 5 cells [prometaphase], n = 440 kinetochores from 5 cells [metaphase]; CENP-C ∆M12BD RPE-1 cells: n = 280 kinetochores from 5 cells [prophase], n = 423 kinetochores from 5 cells [prometaphase], n = 429 kinetochores from 5 cells [metaphase]).

Article Snippet: The primary antibodies included rabbit anti-human Hec1 (1:5,000) (ab3613; Abcam), mouse anti-human CENP-A (1:250) , mouse anti-human Aurora B (1:500) (611082; BD Bioscience), rabbit anti-human DSN1 (1:2,000) (a gift from Iain Cheeseman, Whitehead Institute, MIT) , rabbit anti-human KNL1 (1:2,000) (a gift from Iain Cheeseman, Whitehead Institute, MIT) , mouse anti-human Bub1 (1:400) (MAB3610; Millipore), rabbit anti-H2AT120ph (1:1,000) (Active Motif), mouse anti-BubR1 (1:500) (MAB3612; Millipore), mouse anti-PLK1 (1:500) (ab17057; Abcam), rabbit anti-Ska3 (1:500) (a gift from Gary J Gorbsky) , mouse anti-H3T3ph (1:3,000) , FITC-conjugated mouse anti-α-tubulin (1:1,000) (F2168; Sigma-Aldrich), and mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich).

Techniques: Staining, Marker, Two Tailed Test, Comparison

(A) H2AT120ph localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. H2AT120ph was stained with an antibody against H2AT120ph (green). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 1 μm). H2AT120ph signal intensities at kinetochore-proximal centromeres were quantified. A representative result from three independent experiments is shown (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 316 kinetochores from 5 cells; CENP-T ∆NBD−1 : n = 306 kinetochores from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 325 kinetochores from 5 cells). (B) H3T3ph localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE1 cells. H3T3ph was stained with an antibody against H3T3ph. The insets show an enlarged single chromosome (scale bar, 1 μm). H3T3ph localization at centromeres was examined and quantified. Scale bar, 10 μm. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 195 centromeres from 5 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 206 centromeres from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 210 centromeres from 5 cells). (C) Bub1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE1 cells. Bub1 was stained with an antibody against Bub1 (green). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 427 kinetochores from 5 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 424 kinetochores from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 423 kinetochores from 5 cells).

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) H2AT120ph localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. H2AT120ph was stained with an antibody against H2AT120ph (green). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 1 μm). H2AT120ph signal intensities at kinetochore-proximal centromeres were quantified. A representative result from three independent experiments is shown (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 316 kinetochores from 5 cells; CENP-T ∆NBD−1 : n = 306 kinetochores from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 325 kinetochores from 5 cells). (B) H3T3ph localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE1 cells. H3T3ph was stained with an antibody against H3T3ph. The insets show an enlarged single chromosome (scale bar, 1 μm). H3T3ph localization at centromeres was examined and quantified. Scale bar, 10 μm. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 195 centromeres from 5 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 206 centromeres from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 210 centromeres from 5 cells). (C) Bub1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE1 cells. Bub1 was stained with an antibody against Bub1 (green). mScarlet-CENP-T was used as a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 427 kinetochores from 5 cells; CENP-T ∆NBD−1 RPE-1 cells: n = 424 kinetochores from 5 cells; CENP-T ∆NBD−2 RPE-1 cells: n = 423 kinetochores from 5 cells).

Techniques: Staining, Marker, Comparison

(A) Strategy to generate CENP-T ∆M12BD RPE1 cells. In the cells, mini-auxin-inducible degron (mAID)–tagged human CENP-T was expressed together with OsTIR1 from the AAVS locus. Because mAID-fused CENP-T is degraded upon IAA (indole-3-acetic acid) treatment, mScarlet-tagged CENP-T is only expressed from the endogenous CENP-T locus in CENP-T ∆M12BD RPE1 cells. (B) Schematic representation of mScarlet-CENP-T cDNA targeting the endogenous CENP-T locus. To express mScarlet-tagged Mis12C-binding domain mutant CENP-T (∆M12BD: ∆107-230) or wild-type CENP-T under the control of the endogenous CENP-T promoter, mScarlet-CENP-T cDNAs were targeted into exon 1 by CRISPR/Cas9-mediated homologous recombination. Because the targeting constructs have puromycin resistance genes ( PuroR ) or neomycin resistance genes ( NeoR ), targeted cells were selected using these selection markers. The gRNA sequence and the position of primers for genotyping are shown. (C) Genotyping PCR for targeted mScarlet-CENP-T in CENP-T ∆M12BD RPE1 cells. Genotyping in isolated single clones was performed using the primers shown in (B). WT RPE-1 cells were used as a control. (D) CENP-T protein expression in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. The cells were treated with or without IAA for 2 d, and we examined GFP-mAID-CENP-T and mScarlet-CENP-T protein expression using an antibody against CENP-T or mScarlet (RFP). α-Tubulin was probed as a loading control. (E) Schematic representation of human CENP-T. Human CENP-T WT has two Ndc80C-binding regions and a Mis12-binding domain (M12BD: amino acids 107–230). The M12BD region was deleted in CENP-T ∆M12BD . (F) DSN1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). DSN1 localization at mitotic kinetochores was examined and quantified. mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 10 cells; CENP-T ∆M12BD RPE-1 cells: n = 10 cells. (G) KNL1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). KNL1 localization at mitotic kinetochores was examined and quantified. Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 10 cells; CENP-T ∆M12BD RPE-1 cells: n = 10 cells. (H) Hec1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. Hec1 was stained with an antibody against Hec1 (green). Hec1 localization at mitotic kinetochores was examined and quantified. Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 10 cells; CENP-T ∆M12BD RPE-1 cells: n = 10 cells. (I) Bub1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. Bub1 was stained with an antibody against Bub1 (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 363 kinetochores from 5 cells; CENP-T ∆M12BD RPE-1 cells: n = 346 kinetochores from 5 cells). (J) H2AT120ph localization in CENP-T WT or CENP-T ∆M12BD cells. H2AT120ph was stained with an antibody against H2AT120ph (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. H2AT120ph signal intensities at kinetochore-proximal centromeres were quantified (mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 297 kinetochores from 5 cells; CENP-T ∆M12BD RPE-1 cells: n = 366 kinetochores from 5 cells). (K) Aurora B localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. Aurora B was stained with an antibody against Aurora B (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 1 μm). Aurora B signal intensities at inner centromeres were quantified (mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 186 centromeres from 5 cells; CENP-T ∆M12BD RPE-1 cells: n = 207 centromeres from 5 cells).

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) Strategy to generate CENP-T ∆M12BD RPE1 cells. In the cells, mini-auxin-inducible degron (mAID)–tagged human CENP-T was expressed together with OsTIR1 from the AAVS locus. Because mAID-fused CENP-T is degraded upon IAA (indole-3-acetic acid) treatment, mScarlet-tagged CENP-T is only expressed from the endogenous CENP-T locus in CENP-T ∆M12BD RPE1 cells. (B) Schematic representation of mScarlet-CENP-T cDNA targeting the endogenous CENP-T locus. To express mScarlet-tagged Mis12C-binding domain mutant CENP-T (∆M12BD: ∆107-230) or wild-type CENP-T under the control of the endogenous CENP-T promoter, mScarlet-CENP-T cDNAs were targeted into exon 1 by CRISPR/Cas9-mediated homologous recombination. Because the targeting constructs have puromycin resistance genes ( PuroR ) or neomycin resistance genes ( NeoR ), targeted cells were selected using these selection markers. The gRNA sequence and the position of primers for genotyping are shown. (C) Genotyping PCR for targeted mScarlet-CENP-T in CENP-T ∆M12BD RPE1 cells. Genotyping in isolated single clones was performed using the primers shown in (B). WT RPE-1 cells were used as a control. (D) CENP-T protein expression in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. The cells were treated with or without IAA for 2 d, and we examined GFP-mAID-CENP-T and mScarlet-CENP-T protein expression using an antibody against CENP-T or mScarlet (RFP). α-Tubulin was probed as a loading control. (E) Schematic representation of human CENP-T. Human CENP-T WT has two Ndc80C-binding regions and a Mis12-binding domain (M12BD: amino acids 107–230). The M12BD region was deleted in CENP-T ∆M12BD . (F) DSN1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). DSN1 localization at mitotic kinetochores was examined and quantified. mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 10 cells; CENP-T ∆M12BD RPE-1 cells: n = 10 cells. (G) KNL1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). KNL1 localization at mitotic kinetochores was examined and quantified. Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 10 cells; CENP-T ∆M12BD RPE-1 cells: n = 10 cells. (H) Hec1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. Hec1 was stained with an antibody against Hec1 (green). Hec1 localization at mitotic kinetochores was examined and quantified. Scale bar, 5 μm. Mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 10 cells; CENP-T ∆M12BD RPE-1 cells: n = 10 cells. (I) Bub1 localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. Bub1 was stained with an antibody against Bub1 (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 363 kinetochores from 5 cells; CENP-T ∆M12BD RPE-1 cells: n = 346 kinetochores from 5 cells). (J) H2AT120ph localization in CENP-T WT or CENP-T ∆M12BD cells. H2AT120ph was stained with an antibody against H2AT120ph (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. H2AT120ph signal intensities at kinetochore-proximal centromeres were quantified (mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 297 kinetochores from 5 cells; CENP-T ∆M12BD RPE-1 cells: n = 366 kinetochores from 5 cells). (K) Aurora B localization in CENP-T WT or CENP-T ∆M12BD RPE-1 cells. Aurora B was stained with an antibody against Aurora B (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 1 μm). Aurora B signal intensities at inner centromeres were quantified (mean and SD, two-tailed t test, CENP-T WT RPE-1 cells: n = 186 centromeres from 5 cells; CENP-T ∆M12BD RPE-1 cells: n = 207 centromeres from 5 cells).

Techniques: Binding Assay, Mutagenesis, Control, CRISPR, Homologous Recombination, Construct, Selection, Sequencing, Isolation, Clone Assay, Expressing, Staining, Marker, Two Tailed Test

(A) Strategy to generate RPE-1 cell lines expressing DSN1 WT or DSN1 ∆BM with CENP-C WT or CENP-C ∆M12BD (CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , or CC ∆M12BD /DSN1 ∆BM RPE-1 cells). mScarlet-CENP-A was also expressed in all cell lines. The DSN1 basic motif (amino acids 91–113) was deleted in DSN1 ∆BM . (B) Schematic representation of mScarlet-DSN1 cDNA targeting the endogenous DSN1 locus. To express mScarlet-tagged DSN1 wild-type (WT) or a basic motif deletion mutant (∆BM: ∆91-113) under the control of the endogenous DSN1 promoter, mScarlet-DSN1 WT or ∆BM cDNAs were targeted into the DSN1 locus by CRISPR/Cas9-mediated homologous recombination. Because the targeting constructs have blasticidin S resistance genes ( BsR ), targeted cells were selected using the selection marker. The gRNA sequence and the position of primers for genotyping are shown. (C) Genotyping PCR and immunoblotting for CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , or CC ∆M12BD /DSN1 ∆BM RPE-1 cells. Genotyping in isolated single clones was performed using the primers shown in (B). In immunoblots, DSN1 was detected by an antibody against DSN1 or mScarlet (RFP), and α-tubulin was probed as a loading control. (D) DSN1 localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. DSN1, a subunit of the Mis12C (green), was stained with an antibody against DSN1 (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 10 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 10 cells). (E) KNL1 localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 10 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 10 cells). (F) Bub1 localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. Bub1 was stained with an antibody against Bub1 (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at mitotic kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 354 kinetochores from 5 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 327 kinetochores from 5 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 375 kinetochores from 5 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 386 kinetochores from 5 cells). (G) Aurora B localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. Aurora B was stained with an antibody against Aurora B (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 2 μm). Aurora B signal intensities at inner centromeres were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 228 kinetochores from 5 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 221 kinetochores from 5 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 215 kinetochores from 5 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 212 kinetochores from 5 cells). (H) Aurora B localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells treated with 5-ITu for 30 min. Aurora B was stained with an antibody against Aurora B (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show enlarged chromosomes (scale bar, 2 μm). Aurora B signal intensities at the kinetochore-proximal pool were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 387 kinetochores from 5 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 389 kinetochores from 5 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 381 kinetochores from 5 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 395 kinetochores from 5 cells).

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) Strategy to generate RPE-1 cell lines expressing DSN1 WT or DSN1 ∆BM with CENP-C WT or CENP-C ∆M12BD (CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , or CC ∆M12BD /DSN1 ∆BM RPE-1 cells). mScarlet-CENP-A was also expressed in all cell lines. The DSN1 basic motif (amino acids 91–113) was deleted in DSN1 ∆BM . (B) Schematic representation of mScarlet-DSN1 cDNA targeting the endogenous DSN1 locus. To express mScarlet-tagged DSN1 wild-type (WT) or a basic motif deletion mutant (∆BM: ∆91-113) under the control of the endogenous DSN1 promoter, mScarlet-DSN1 WT or ∆BM cDNAs were targeted into the DSN1 locus by CRISPR/Cas9-mediated homologous recombination. Because the targeting constructs have blasticidin S resistance genes ( BsR ), targeted cells were selected using the selection marker. The gRNA sequence and the position of primers for genotyping are shown. (C) Genotyping PCR and immunoblotting for CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , or CC ∆M12BD /DSN1 ∆BM RPE-1 cells. Genotyping in isolated single clones was performed using the primers shown in (B). In immunoblots, DSN1 was detected by an antibody against DSN1 or mScarlet (RFP), and α-tubulin was probed as a loading control. (D) DSN1 localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. DSN1, a subunit of the Mis12C (green), was stained with an antibody against DSN1 (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 10 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 10 cells). (E) KNL1 localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 10 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 10 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 10 cells). (F) Bub1 localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. Bub1 was stained with an antibody against Bub1 (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at mitotic kinetochores were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 354 kinetochores from 5 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 327 kinetochores from 5 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 375 kinetochores from 5 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 386 kinetochores from 5 cells). (G) Aurora B localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells. Aurora B was stained with an antibody against Aurora B (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 2 μm). Aurora B signal intensities at inner centromeres were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 228 kinetochores from 5 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 221 kinetochores from 5 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 215 kinetochores from 5 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 212 kinetochores from 5 cells). (H) Aurora B localization in CC WT /DSN1 WT , CC WT /DSN1 ∆BM , CC ∆M12BD /DSN1 WT , and CC ∆M12BD /DSN1 ∆BM RPE-1 cells treated with 5-ITu for 30 min. Aurora B was stained with an antibody against Aurora B (green). The mScarlet-CENP-A and mScarlet-DSN1 were used as a kinetochore marker (CENP-A/DSN1, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show enlarged chromosomes (scale bar, 2 μm). Aurora B signal intensities at the kinetochore-proximal pool were quantified. A representative result from two independent experiments is shown (mean and SD, one-way ANOVA with Tukey’s multiple comparison test, CC WT /DSN1 WT RPE-1 cells: n = 387 kinetochores from 5 cells; CC WT /DSN1 ∆BM RPE-1 cells: n = 389 kinetochores from 5 cells; CC ∆M12BD /DSN1 WT RPE-1 cells: n = 381 kinetochores from 5 cells; CC ∆M12BD /DSN1 ∆BM RPE-1 cells: n = 395 kinetochores from 5 cells).

Techniques: Expressing, Mutagenesis, Control, CRISPR, Homologous Recombination, Construct, Selection, Marker, Sequencing, Western Blot, Isolation, Clone Assay, Staining, Comparison

(A) Schematic representation for forced binding of Mis12C to CENP-C in HeLa cells. To validate the idea that the CENP-C-Mis12C interaction positively regulates Aurora B localization, we used HeLa cells in which Aurora B activity is low at centromeres, leading to chromosome instability. The CENP-C-Mis12C interaction was increased by expressing a DSN1 mutant lacking the basic motif (∆BM) in HeLa cells, and we examined the Aurora B levels and efficiency of kinetochore–microtubule error correction. Dsn1 is a subunit of Mis12C. The basic motif of DSN1 masks the CENP-C-binding surface of Mis12C, preventing the CENP-C-Mis12C interaction. The deletion of the basic motif increases the binding affinity between CENP-C and Mis12C . (B) DSN1 localization in DSN1 WT or DSN1 ∆BM HeLa cells. DSN1, a subunit of the Mis12 complex, was stained with an antibody against DSN1 (red). DNA was stained with DAPI (blue). CENP-A was stained by an antibody against CENP-A as a kinetochore marker (green). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified (mean and SD, two-tailed t test, DSN1 WT HeLa cells: n = 10 cells; DSN1 ∆BM HeLa cells: n = 10 cells). (C) Bub1 localization in DSN1 WT or DSN1 ∆BM HeLa cells. Bub1 was stained with an antibody against Bub1 (green). DSN1 was stained as a kinetochore marker (red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, DSN1 WT HeLa cells: n = 523 kinetochores from 6 cells; DSN1 ∆BM HeLa cells: n = 501 kinetochores from 6 cells). (D) Aurora B and H2AT120ph localization in DSN1 WT or DSN1 ∆BM HeLa cells. Aurora B and H2AT120ph were stained with their antibodies (green and cyan). mScarlet-DSN1 was used as a kinetochore marker (DSN1, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 2 μm). The signal intensities of Aurora B at centromeres and H2AT120ph at kinetochore-proximal centromeres were quantified (mean and SD, two-tailed t test, DSN1 WT HeLa cells: n = 385 centromeres, 651 kinetochores from 6 cells for Aurora B and H2AT120ph, respectively; DSN1 ∆BM HeLa cells: n = 379 centromeres, 668 kinetochores from 6 cells for Aurora B and H2AT120ph, respectively). (E) Error correction assay in DSN1 WT or DSN1 ∆BM HeLa cells. The cells were treated with monastrol for 4 h and then released and incubated in a medium with MG132 for 30 or 45 min. The cells were fixed, and microtubules (green) and DNA (blue) were stained with an antibody against alpha-tubulin and DAPI, respectively. mScarlet-DSN1 was used as a kinetochore marker (DSN1, red). The cells with more than two unaligned chromosomes were defined as “cells with unaligned chromosomes.” Arrows indicate unaligned chromosomes. Scale bar, 5 μm. Mitotic cells with unaligned chromosomes were quantified. Four independent experiments were performed (mean and SD, two-tailed t test). (F) Model for a positive regulatory loop to facilitate Aurora B localization at the centromere through the CENP-C-Mis12C interaction. The regulatory system is required for efficient error correction of kinetochore–microtubule attachment, leading to accurate chromosome segregation.

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 for forced binding of Mis12C to CENP-C in HeLa cells. To validate the idea that the CENP-C-Mis12C interaction positively regulates Aurora B localization, we used HeLa cells in which Aurora B activity is low at centromeres, leading to chromosome instability. The CENP-C-Mis12C interaction was increased by expressing a DSN1 mutant lacking the basic motif (∆BM) in HeLa cells, and we examined the Aurora B levels and efficiency of kinetochore–microtubule error correction. Dsn1 is a subunit of Mis12C. The basic motif of DSN1 masks the CENP-C-binding surface of Mis12C, preventing the CENP-C-Mis12C interaction. The deletion of the basic motif increases the binding affinity between CENP-C and Mis12C . (B) DSN1 localization in DSN1 WT or DSN1 ∆BM HeLa cells. DSN1, a subunit of the Mis12 complex, was stained with an antibody against DSN1 (red). DNA was stained with DAPI (blue). CENP-A was stained by an antibody against CENP-A as a kinetochore marker (green). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified (mean and SD, two-tailed t test, DSN1 WT HeLa cells: n = 10 cells; DSN1 ∆BM HeLa cells: n = 10 cells). (C) Bub1 localization in DSN1 WT or DSN1 ∆BM HeLa cells. Bub1 was stained with an antibody against Bub1 (green). DSN1 was stained as a kinetochore marker (red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, DSN1 WT HeLa cells: n = 523 kinetochores from 6 cells; DSN1 ∆BM HeLa cells: n = 501 kinetochores from 6 cells). (D) Aurora B and H2AT120ph localization in DSN1 WT or DSN1 ∆BM HeLa cells. Aurora B and H2AT120ph were stained with their antibodies (green and cyan). mScarlet-DSN1 was used as a kinetochore marker (DSN1, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (scale bar, 2 μm). The signal intensities of Aurora B at centromeres and H2AT120ph at kinetochore-proximal centromeres were quantified (mean and SD, two-tailed t test, DSN1 WT HeLa cells: n = 385 centromeres, 651 kinetochores from 6 cells for Aurora B and H2AT120ph, respectively; DSN1 ∆BM HeLa cells: n = 379 centromeres, 668 kinetochores from 6 cells for Aurora B and H2AT120ph, respectively). (E) Error correction assay in DSN1 WT or DSN1 ∆BM HeLa cells. The cells were treated with monastrol for 4 h and then released and incubated in a medium with MG132 for 30 or 45 min. The cells were fixed, and microtubules (green) and DNA (blue) were stained with an antibody against alpha-tubulin and DAPI, respectively. mScarlet-DSN1 was used as a kinetochore marker (DSN1, red). The cells with more than two unaligned chromosomes were defined as “cells with unaligned chromosomes.” Arrows indicate unaligned chromosomes. Scale bar, 5 μm. Mitotic cells with unaligned chromosomes were quantified. Four independent experiments were performed (mean and SD, two-tailed t test). (F) Model for a positive regulatory loop to facilitate Aurora B localization at the centromere through the CENP-C-Mis12C interaction. The regulatory system is required for efficient error correction of kinetochore–microtubule attachment, leading to accurate chromosome segregation.

Techniques: Binding Assay, Activity Assay, Expressing, Mutagenesis, Staining, Marker, Two Tailed Test, Incubation

Analysis of BUB1 and BUB1B gene differential expression in tumors. ( A ) Analysis of BUB1 gene expression in tumors in TIMER 2.0 database; ( B ) Analysis of BUB1 gene expression analysis in tumors in GEPIA2 database; ( C ) Analysis of BUB1B gene expression analysis in tumors in TIMER 2.0 database; ( D ) Analysis of BUB1B gene expression analysis in tumors in GEPIA2 database. * P < 0.05.

Journal: Scientific Reports

Article Title: Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

doi: 10.1038/s41598-024-67528-2

Figure Lengend Snippet: Analysis of BUB1 and BUB1B gene differential expression in tumors. ( A ) Analysis of BUB1 gene expression in tumors in TIMER 2.0 database; ( B ) Analysis of BUB1 gene expression analysis in tumors in GEPIA2 database; ( C ) Analysis of BUB1B gene expression analysis in tumors in TIMER 2.0 database; ( D ) Analysis of BUB1B gene expression analysis in tumors in GEPIA2 database. * P < 0.05.

Article Snippet: Samples were incubated with primary antibody (rabbit anti-human BUB1 and BUBIB monoclonal antibody, 1:200) (Sanying Biotechnology Co. Ltd., Wuhan, Hubei, China), followed by incubation with secondary antibody (HRP-labeled goat anti-rabbit II antibody, 1:200) (Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China).

Techniques: Quantitative Proteomics, Gene Expression

BUB1 and BUB1B expression in EC. ( A ) BUB1 expression in normal endometrium and EC (XianTao database); ( B ) BUB1 expression in normal endometrium and EC (IHC); ( C ) BUB1B expression in normal endometrium and EC (XiaoTao database); ( D ) BUB1B expression in normal endometrium and EC (IHC); ( E ) BUB1 ROC curve in EC; ( F ) BUB1B ROC curve in EC.

Journal: Scientific Reports

Article Title: Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

doi: 10.1038/s41598-024-67528-2

Figure Lengend Snippet: BUB1 and BUB1B expression in EC. ( A ) BUB1 expression in normal endometrium and EC (XianTao database); ( B ) BUB1 expression in normal endometrium and EC (IHC); ( C ) BUB1B expression in normal endometrium and EC (XiaoTao database); ( D ) BUB1B expression in normal endometrium and EC (IHC); ( E ) BUB1 ROC curve in EC; ( F ) BUB1B ROC curve in EC.

Techniques: Expressing

BUB1 and BUB1B gene variations in EC. ( A ) BUB1 gene alteration in EC; ( B ) BUB1 gene alteration in relation to OS in EC; ( C ) BUB1 gene alteration in relation to PFS in EC. ( D ) BUB1B gene alteration in EC; € BUB1B gene alteration in relation to OS in EC; F. BUB1B gene alteration in relation to PFS in EC.

Journal: Scientific Reports

Article Title: Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

doi: 10.1038/s41598-024-67528-2

Figure Lengend Snippet: BUB1 and BUB1B gene variations in EC. ( A ) BUB1 gene alteration in EC; ( B ) BUB1 gene alteration in relation to OS in EC; ( C ) BUB1 gene alteration in relation to PFS in EC. ( D ) BUB1B gene alteration in EC; € BUB1B gene alteration in relation to OS in EC; F. BUB1B gene alteration in relation to PFS in EC.

Techniques:

BUB1 or BUB1B and EC immune filtration. ( A ) BUB1 expression was associated with TIL abundance in EC; ( B ) BUB1 expression was associated with immunosuppressants in EC; ( C ) BUB1 expression and immunostimulants in EC. ( D ) BUB1B expression was associated with TIL abundance in EC; E. BUB1B expression was associated with immunosuppressants in EC; ( F ) BUB1B expression and immunostimulants in EC.

Journal: Scientific Reports

Article Title: Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

doi: 10.1038/s41598-024-67528-2

Figure Lengend Snippet: BUB1 or BUB1B and EC immune filtration. ( A ) BUB1 expression was associated with TIL abundance in EC; ( B ) BUB1 expression was associated with immunosuppressants in EC; ( C ) BUB1 expression and immunostimulants in EC. ( D ) BUB1B expression was associated with TIL abundance in EC; E. BUB1B expression was associated with immunosuppressants in EC; ( F ) BUB1B expression and immunostimulants in EC.

Techniques: Filtration, Expressing

The relation between BUB1 and BUB1B expression with clinicopathological characteristics in EC.

Journal: Scientific Reports

Article Title: Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

doi: 10.1038/s41598-024-67528-2

Figure Lengend Snippet: The relation between BUB1 and BUB1B expression with clinicopathological characteristics in EC.

Techniques: Expressing

Influence of BUB1 and BUBIB knockdown on EC cell phenotype. ( A ) The growth of Ishikawa cells in each group; ( B ) The migration rates of Ishikawa cells in each group: blank group (50.99 ± 1.02)%, NC-siRNA group (66.50 ± 7.74)%, BUB1-siRNA group (34.87 ± 4.26)%, and BUB1B-siRNA group (31.96 ± 4.72)%; ( C ) The number of cells that crossed the membrane in each group: blank group (127.3 ± 4.8), NC-siRNA group (125.3 ± 3.9), BUB1-siRNA group (48.6 ± 3.2), and BUB1B-siRNA group (35.0 ± 2.1). * P < 0.05, ** P < 0.01, *** P < 0.001.

Journal: Scientific Reports

Article Title: Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

doi: 10.1038/s41598-024-67528-2

Figure Lengend Snippet: Influence of BUB1 and BUBIB knockdown on EC cell phenotype. ( A ) The growth of Ishikawa cells in each group; ( B ) The migration rates of Ishikawa cells in each group: blank group (50.99 ± 1.02)%, NC-siRNA group (66.50 ± 7.74)%, BUB1-siRNA group (34.87 ± 4.26)%, and BUB1B-siRNA group (31.96 ± 4.72)%; ( C ) The number of cells that crossed the membrane in each group: blank group (127.3 ± 4.8), NC-siRNA group (125.3 ± 3.9), BUB1-siRNA group (48.6 ± 3.2), and BUB1B-siRNA group (35.0 ± 2.1). * P < 0.05, ** P < 0.01, *** P < 0.001.

Techniques: Knockdown, Migration, Membrane

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