Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 1 | Recruitment of Cdc20 to kinetochores requires BubR1. (a) Schematic of protocol used to synchronize cells and deplete specific proteins using RNAi. (b) Cells depleted of the indicated proteins were stained for CRESTand Cdc20 and BubR1 or Mad2 to check for depletion efficiency. Scale bar, 5 mm. (c) Quantification of Cdc20, BubR1 and Mad2 kinetochore levels in cells treated with the indicated RNAi oligos. The fluorescence from the three z-stacks (200 nm apart) encompassing the bulk kinetochore fluorescent intensity was used and normalized to the CREST signal. Cells were co-stained for Mad2 or BubR1 to ensure that only cells with efficient depletion were analysed. At least 80 kinetochore pairs from eight cells were quantified and the mean and s.e.m. is indicated. (d) Stable HeLa cell line expressing Venus-Cdc20 was treated with the indicated RNAi oligos and the localization of Venus-Cdc20 followed by time-lapse microscopy. Scale bar, 10 mm. (e) Quantification of Venus-Cdc20 at kinetochores in the movies in (d). The signal was quantified from a single z-section and in the frame right after NEBD. A total number of 50 kinetochores from 10 cells were analyzed for each condition and the mean and s.e.m. indicated. An unpaired t-test was performed for statistical analysis (****Po0.0001) and compared with the control-treated cells.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Staining, Expressing, Time-lapse Microscopy, Control

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 3 | The internal Cdc20 binding site of BubR1 recruits Cdc20 to kinetochores. (a) Schematic of human BubR1 and the location of Cdc20 binding sites and Bub3 binding site as well as the pseudo-kinase domain. Alignment of the region encompassing residues 530–535 of human BubR1 is shown on top and the different constructs used are indicated below. The BubR1 KEN/AAA has the first KEN-box mutated to AAA. (b) Stable HeLa cell lines expressing the different Venus-BubR1 siRNA resistant constructs were used to determine the domains in BubR1 required for Cdc20 kinetochore localization. In brief, cells were treated with a control RNAi oligo (Luciferase) or a BubR1 RNAi oligo and then arrested in mitosis using nocodazole treatment and the proteasome inhibitor, MG132. BubR1 RNAi-treated cells were complemented with the indicated Venus-BubR1 constructs. Cells were stained for BubR1, CREST and Cdc20. Scale bar, 5 mm (c,d). The kinetochore levels of BubR1 (c) and Cdc20 (d) normalized to CREST in control and BubR1-depleted cells. At least 80 kinetochore pairs from eight different cells were analysed, and the mean and s.e.m. are indicated. (e) The level of Cdc20 at kinetochores in cells complemented with the indicated Venus-BubR1 constructs was determined. Only cells with endogenous levels of BubR1 at kinetochores were used for this analysis. At least 80 kinetochore pairs from eight different cells were analysed, and the mean and s.e.m. are indicated. (f) Binding of Cdc20 and Cdc20 4A to BubR1 516–715 was determined by binding 10 mg recombinant FLAG-HA-BubR1 516–715 to FLAG affinity beads and incubating these with increasing concentrations of Strep-His tagged Cdc20 or Cdc20 4A expressed and purified from HEK293 cells. The beads were washed and bound proteins were eluted and analysed by western blot. (g) Quantification of western blot in (f) using Licor technology. Experiment in (f,g) is representative of two independent experiments.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Binding Assay, Construct, Expressing, Control, Luciferase, Staining, Recombinant, Western Blot

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 5 | The IC20BD is required for SAC silencing. (a) Stable HeLa cells were depleted of endogenous BubR1 or treated with control RNAi oligo (Luciferase). BubR1 knockdown was complemented by expression of Venus-BubR1, Venus-BubR1 DBub3 or Venus-BubR1 DBub3/D490–560 as indicated and mitotic progression was followed by time-lapse microscopy. Each circle represents a single cell analysed (at least 40 cells were analysed for each condition) and the median (m) is shown as a red bar. Statistical analysis was performed using a Mann–Whitney test. (b) Stable HeLa cell lines were depleted of endogenous BubR1 and complemented by expression of Venus-BubR1 or Venus-BubR1 D490–560 as indicated. Cells were arrested in mitosis with 100 nM nocodazole before filming and subsequently treated with 0.5 mM reversine to silence the SAC. Each circle represents a single cell analysed and at least 120 cells were analysed per condition. Red bars indicate means (m) and a t-test was used for statistical analysis. (c) Representative still images from (b). Scale bar, 10 mm. (d,e) Venus-Cdc20 was purified from a stable HeLa cell line arrested in mitosis with nocodazole. The beads were incubated with increasing concentrations of recombinant FLAG-HA-BubR1 516–715 at room temperature for 1 h and afterwards washed. The binding of endogenous BubR1 and Mad2 to Venus-Cdc20 was analysed by western blot analysis and quantified using Licor technology. Representative of two independent experiments. (f) HeLa cells were transfected with the indicated BubR1 constructs and before filming 200 nM taxol was added. The time from NEBD to mitotic exit was measured by analysing the time-lapse movies and at least 50 cells were analysed per condition. Medians (m) are shown as red bars and a Mann–Whitney test was used for statistical analysis.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Control, Luciferase, Knockdown, Expressing, Time-lapse Microscopy, MANN-WHITNEY, Incubation, Recombinant, Binding Assay, Western Blot, Transfection, Construct

Journal: Oncogene

Article Title: BubR1 is involved in regulation of DNA damage responses.

doi: 10.1038/sj.onc.1209392

Figure Lengend Snippet: Figure 1 Compromised cell cycle G2/M arrest of BubR1 þ / MEFs after DNA damage. (a) BubR1 þ / þ MEFs, BubR1 þ / MEFs, and HeLa cells were treated with adriamycin (ADR, 0.5 mM) for various intervals as indicated (h). Treated cells were then collected, fixed, stained with DAPI, and processed for cell-cycle analysis by flow cytometry. These paired MEFs were in their third passage. (b) BubR1 þ / þ and BubR1 þ / MEFs of the third passage were treated with nocodazole for 16 h. The cells were then released from nocodazole block and cultured in the presence of doxorubicin for various times, as indicated. Cells were fixed, stained, and processed for cell-cycle analysis by flow cytometry. (c) BubR1 þ / þ and BubR1 þ / MEFs of the third passage were treated with nocodazole for 16 h. The cells were then released from nocodazole block and cultured in the presence or absence of doxorubicin for various times. The percent of G2/M cells were calculated. These experi- ments were repeated for at least two times with independent lines of primary MEFs and representative data were presented.

Article Snippet: After preclearing with protein A/G beads (Santa Cruz), protein lysates of interphase and mitotic stages (10mg each) were incubated with anti-BubR1 antibody (40 mg) overnight at 41C.

Techniques: Staining, Cell Cycle Assay, Cytometry, Blocking Assay, Cell Culture

Journal: Oncogene

Article Title: BubR1 is involved in regulation of DNA damage responses.

doi: 10.1038/sj.onc.1209392

Figure Lengend Snippet: Figure 2 Weakened DNA damage responses in BubR1 þ / MEFs. (a) BubR1 þ / þ (W, wild type) and BubR1 þ / (H, heterozygous) MEFs were treated with doxorubicin (Dox) for various times. An equal amount of cell lysates was analysed for phospho-histone H2AX (p-H2AX) and b-actin by Western blotting. (b) BubR1 þ / þ (W) and BubR1 þ / (H) MEFs were exposed to UV irradiation. At various time intervals post irradiation, cells were collected for lysis and equal amounts of cell lysates were blotted for p-H2AX and b- actin. (c) BubR1 þ / þ and BubR1 þ / MEFs were treated with doxorubicin (0.5 mM) for various time intervals. Equal amounts of cell lysates were blotted for p53, p21, and b-actin. (d) BubR1 þ / þ and BubR1 þ / MEFs exposed to UV irradiation for various times were lysed and equal amounts of cell lysates were blotted for p53, p21 and b-actin. The MEFs used in this series of experiments were in their fourth passage. Each experiment was repeated for at least three times. Representative results were shown.

Article Snippet: After preclearing with protein A/G beads (Santa Cruz), protein lysates of interphase and mitotic stages (10mg each) were incubated with anti-BubR1 antibody (40 mg) overnight at 41C.

Techniques: Western Blot, Irradiation, Lysis

Journal: Oncogene

Article Title: BubR1 is involved in regulation of DNA damage responses.

doi: 10.1038/sj.onc.1209392

Figure Lengend Snippet: Figure 3 BubR1 deficiency results in downregulation of p21, correlating with a better survival rate. (a) HeLa cells were transfected with BubR1 or luciferase (Luc) siRNA duplex for 48 h. Equal amounts of cell lysates from the transfected cells, as well as from the non-transfected controls cells, were blotted for p21, securin, cyclin B1, and b-actin. (b) Human 293 cells were transfected with BubR1 or luciferase (Luc) siRNA duplex for 48 h. Equal amounts of cell lysates from the transfected cells, as well as from the non-transfected controls cells, were blotted for p53, p21, and b-actin. Each experiment was repeated for at least two times with independent lines of primary MEFs. Representative results were shown. (c) BubR1 þ / þ and BubR1 þ / MEFs of the fourth passage were treated with various concentrations of doxorubicin, as indicated. At 24 h after treatment with the DNA damage agent, the cell viability was measured by the MTT assay. The data were summarized from three independent experiments.

Article Snippet: After preclearing with protein A/G beads (Santa Cruz), protein lysates of interphase and mitotic stages (10mg each) were incubated with anti-BubR1 antibody (40 mg) overnight at 41C.

Techniques: Transfection, Luciferase, MTT Assay

Journal: Oncogene

Article Title: BubR1 is involved in regulation of DNA damage responses.

doi: 10.1038/sj.onc.1209392

Figure Lengend Snippet: Figure 4 Genotoxic stresses induce BubR1 degradation. (a) BubR1 þ / þ and BubR1 þ / MEFs were treated with doxorubicin for various times as indicated. Cell lysates from various treatments were blotted for BubR1 and actin. (b) HeLa cells were treated with doxorubicin for various times and cell lysates were blotted for BubR1 and Mad2. (c) BubR1 þ / þ and BubR1 þ / MEFs were treated with UV for various times. Cell lysates from various treatments were blotted for BubR1 and actin. Phosphorylated BubR1 was indicated as p-BubR1. MEFs used were in their fourth passage.

Article Snippet: After preclearing with protein A/G beads (Santa Cruz), protein lysates of interphase and mitotic stages (10mg each) were incubated with anti-BubR1 antibody (40 mg) overnight at 41C.

Techniques:

Journal: Oncogene

Article Title: BubR1 is involved in regulation of DNA damage responses.

doi: 10.1038/sj.onc.1209392

Figure Lengend Snippet: Figure 5 PARP-1 interacts with BubR1. (a) Mitotic or interphase cell lysates were immunoprecipitated with BubR1 or preimmune IgGs. Immunoprecipitates were thoroughly washed and analysed by SDS-PAGE followed by silver staining. Mitotic and interphase lysates were also blotted for confirmation of the position of phospho- and non-phospho-BubR1 bands in immunoprecipitates. Arrows denote some of the silver-stained bands whose identities were revealed by mass spectrometric analysis. (b) Mitotic (M) and interphase (I) cell lysates were immunoprecipitated with PARP-1 IgGs or with control IgGs. Immunoprecipitates, along with cell lysates from mitotic and interphase cells, were then blotted for BubR1. (c) Mitotic (M) and interphase (I) cell lysates were immunoprecipitated with BubR1 IgGs. Immunoprecipitates as well as cell lysates were blotted for PARP-1.

Article Snippet: After preclearing with protein A/G beads (Santa Cruz), protein lysates of interphase and mitotic stages (10mg each) were incubated with anti-BubR1 antibody (40 mg) overnight at 41C.

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Staining, Control

Journal: Oncogene

Article Title: BubR1 is involved in regulation of DNA damage responses.

doi: 10.1038/sj.onc.1209392

Figure Lengend Snippet: Figure 6 Functional interaction between BubR1 and PARP-1. (a) HeLa cells cultured on chamber slides were stained with antibodies to BubR1 (red) and PARP-1 (green). DNA was stained with DAPI (blue). Indirect immunofluorescence microscopy was performed. A representative prophase cell was shown. (b) BubR1 þ / þ and BubR1 þ / MEFs of the fourth passage were treated with doxorubicin for various times. Equal amounts of lysates from the treated cells, as well as from untreated control cells, were blotted for PARP-1 and b-actin. The position of p89, the major degradation product of PARP-1 was also indicated. (c) BubR1 þ / þ and BubR1 þ / MEFs of the fourth passage were exposed to UV irradiation. At 24 h post UV exposure, cells were collected and lysed. Equal amounts of control and treated cell lysates were blotted for PARP-1, p89, and b-actin. These experiments were repeated with three independent lines of primary MEFs (in the third or fourth passage) and similar results were obtained. (d) HeLa cells were transfected with BubR1 or luciferease (Luc) siRNA for 2 days after which cells were lysed and equal amounts of cell lysates in duplicates were blotted for BubR1, PARP-1, and b-actin.

Article Snippet: After preclearing with protein A/G beads (Santa Cruz), protein lysates of interphase and mitotic stages (10mg each) were incubated with anti-BubR1 antibody (40 mg) overnight at 41C.

Techniques: Functional Assay, Cell Culture, Staining, Microscopy, Control, Irradiation, Transfection

Journal: Molecular Biology of the Cell

Article Title: Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis

doi: 10.1091/mbc.E12-05-0371

Figure Lengend Snippet: p110β knockdown reduces p-Aurora B activity in kinetochores. (A) Control, p110α, or p110β knocked-down U2OS cells (48 h) were monastrol-treated (100 μM, 4 h) and released in medium (60 min). Representative confocal z -sections of IF performed with the indicated antibodies and Hoechst 33258. Graphs show the percentage of p-Aurora B–positive KT in prometaphase. (B) U2OS cells were transfected with control or p110β siRNA, synchronized with monastrol (100 μM, 4 h), and collected after monastrol deprivation at 10 or 45 min. Total cell extracts and soluble and chromatin fractions were tested by Western blotting. Graphs show the p-Aurora B/Aurora B ratio in the chromatin fraction (left). (C) p110β knocked-down U2OS cells (48 h) were monastrol-treated (4 h) and then released (1 h); cells were stained with the indicated Ab. Representative confocal sections of metaphase cells with unaligned chromatids (indicated with an arrow). (D) U2OS cells were transfected as in (A) and maintained in monastrol for 7 h. Representative confocal z -stacks of IF performed with indicated antibodies and Hoechst 33258. We measured the fluorescence intensity of BubR1 signal in arbitrary units (AU, pixels) for each KT (each dot represents a single KT; n = 3 cells of each type). We also calculated the mean fluorescence intensity per cell and represented (right) the mean ± SD of these values for each cell type ( n = 15 cells). Scale bar: 5 μm. Student's t test: **, p < 0.01; *, p < 0.05 (A and D, Student's test with Welch´s correction).

Article Snippet: For immunoprecipitation, we used anti-p110α, anti-p110β, anti-p150Glued, and anti-p85; for immunofluorescence assays, anti- α -tubulin, anti-PIP 3 (Echelon Biosciences, Salt Lake City, UT), anti-CenpA ( Kremer et al. , 1988 ; Abcam), anti-p150Glued, anti-Mad2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p110α, anti-p110β, anti-Myc (Cell Signaling), anti-centromere antigen (anti-ACA; Antibodies Incorporated, Davis, CA), anti-CenpA, anti–phospho-Thr323-Aurora B and anti–Aurora B. BubR1 Ab was from Novus Biologicals (Littleton, CO); Mad1 and Bub1 antibodies were kindly donated by E. Nigg (Biozentrum, University of Basel, Switzerland).

Techniques: Knockdown, Activity Assay, Control, Transfection, Western Blot, Staining, Fluorescence

Journal: Nature cell biology

Article Title: Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

doi: 10.1038/ncb2643

Figure Lengend Snippet: Increased BubR1 expression protects against spontaneous tumors and extends lifespan. (a) Survival curves of wildtype, T-GFP, and T23 mice dying of cancer. Only mice with malignant lymphomas, sarcomas and carcinomas are included. Statistical analysis of the survival curves is represented by the asterisks (log-rank test). (b) Overall survival curves of wildtype, T-GFP, and T23 mice. We note that maximum lifespan of T23 mice was also significantly extended compared to both wildtype and T-GFP control mice (p = 0.05 and 0.0056), respectively; one-sided Wang/Allison test referring to the proportion of mice alive at the 90 th percentile survival point. Furthermore, we note that the median lifespan of our wildtype cohort is similar to that of an earlier, independent study performed at the same site . * P < 0.05, ** P < 0.01, *** P < 0.001. n indicates the number of mice (mixed gender) per genotype.

Article Snippet: Blots were probed with antibodies for P-H3 Ser10 (Millipore; 06-570, 1:500), BubR1 (1:1000) Flag (Origene; TA100011, 1:1000), β-actin (Sigma; A5441, 1:40000), EGFP (Cell Signaling; 2956, 1:1000), Aurora B (BD Biosciences; 611083, 1:1000), Cdc20 (1:1000), Plk1 (1:500) and Hras (1:5000) (Santa Cruz Biotechnology, Inc.; SC-8358, SC-17783, and SC-520, respectively), Bub3 (1:1000), Mad2 (1:1000), and Bub1 (1:1000).

Techniques: Expressing

Journal: Nature cell biology

Article Title: Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

doi: 10.1038/ncb2643

Figure Lengend Snippet: Resistance to tumorigenesis and chromosomal instability in BubR1 transgenic mice. (a) Tumor incidence and multiplicity in animals treated with DMBA on dorsal skin on post-natal day 5 and biopsied at 5 months of age. Values represent means ± SEM. (b) Resistance of BubR1 transgenic mice to lung tumors induced by oncogenic Kras (G12D). Cohorts of Kras LA1 and T23; Kras LA1 mice were killed at 6 weeks of age and lung tumors counted under a dissection microscope. Values represent means ± SD. (c) Western blots of cell extracts from wildtype and T23 MEFs with and without retrovirally expressed oncogenic Hras (G12V). Actin was used as a loading control. (d and e) Oncogenic Hras-induced aneuploidy (d) and chromosome missegregation (e) rates of MEF lines with and without BubR1 overexpression (n = 3 independent MEF lines each). Values represent means ± SD in d and means ± SEM in e . (f) Aneuploidy rates in lungs of wildtype and Kras LA1 mice with and without BubR1 overexpression. n = 3 mice for WT and T23, and n = 4 for Ras and T23;Ras. Values represent means ± SEM. (g) Nocodazole-challenge assay showing that elevated BubR1 expression restores normal mitotic checkpoint activity in MEFs with low amounts of Rae1. Values represent means ± SD. Three independent MEF lines per genotype were used. (h) Aneuploidy rates of mutant MEF lines with and without BubR1 overexpression (n = 5 MEF lines each). Values represent means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. n indicates the number of mice (mixed gender) used per genotype in a and b .

Article Snippet: Blots were probed with antibodies for P-H3 Ser10 (Millipore; 06-570, 1:500), BubR1 (1:1000) Flag (Origene; TA100011, 1:1000), β-actin (Sigma; A5441, 1:40000), EGFP (Cell Signaling; 2956, 1:1000), Aurora B (BD Biosciences; 611083, 1:1000), Cdc20 (1:1000), Plk1 (1:500) and Hras (1:5000) (Santa Cruz Biotechnology, Inc.; SC-8358, SC-17783, and SC-520, respectively), Bub3 (1:1000), Mad2 (1:1000), and Bub1 (1:1000).

Techniques: Transgenic Assay, Dissection, Microscopy, Western Blot, Over Expression, Expressing, Activity Assay, Mutagenesis

Journal: Nature cell biology

Article Title: Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

doi: 10.1038/ncb2643

Figure Lengend Snippet: Increased BubR1 expression delays select age-related pathologies. (a) Sustained BubR1 expression attenuates muscle fiber atrophy. Gastrocnemius muscle fiber diameter declines with age in all genotypes but T23. (b) Quantitative RT-PCR analysis of p16 Ink4a expression in gastrocnemius muscles 3 and 24-month-old transgenic and control mice relative to 3-month-old wildtype muscles. (c) Same as (b) but for p19 Arf . (d–f) Exercise ability is enhanced in aged T23 mice. Time (d), distance (e), and work performed (f) are all improved in 18-month-old T23 animals compared to WT and T-GFP. (g) Hematoxylin-eosin stained kidney sections of 24-month-old mice. Dashed area depicts interstitial inflammation; arrows denote glomerular hypercellularity of WT mice (above) and normal glomeruli of T23 mice (below). Scale bar, 100 μm. (h) Percentage of sclerotic glomeruli from 24-month-old kidney sections. 40 glomeruli were scored for each animal. (i) DNA damage, as measured by γ-H2AX staining on cryosections of kidney tissue, is increased in BubR1 H/H mice at a young age and reduced in transgenic mice at advanced age. (j) Cardiac stress tolerance correlates with BubR1 level of expression. (k) Ptah stained heart sections of 24-month-old wildtype and T23 mice. Interstitial fibrosis (pink area) is significantly reduced in T23 animals. Scale bar, 50 μm. For all analyses in a–k, n = 5 males per genotype per age group. Error bars represent SD. (l) Quantification of the percentage of stem cells isolated from 3 and 24-month-old wildtype and T23 mouse skeletal muscle (satellite cells) and heart (cardiac stem cells). n = 3 males per genotype per tissue. Values represent means ± SEM. We note that there are no significant differences between wildtype and T23. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Blots were probed with antibodies for P-H3 Ser10 (Millipore; 06-570, 1:500), BubR1 (1:1000) Flag (Origene; TA100011, 1:1000), β-actin (Sigma; A5441, 1:40000), EGFP (Cell Signaling; 2956, 1:1000), Aurora B (BD Biosciences; 611083, 1:1000), Cdc20 (1:1000), Plk1 (1:500) and Hras (1:5000) (Santa Cruz Biotechnology, Inc.; SC-8358, SC-17783, and SC-520, respectively), Bub3 (1:1000), Mad2 (1:1000), and Bub1 (1:1000).

Techniques: Expressing, Quantitative RT-PCR, Transgenic Assay, Staining, Isolation

Journal: Nature cell biology

Article Title: Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

doi: 10.1038/ncb2643

Figure Lengend Snippet: Transgenic mouse strains with moderate and high levels of Flag-BubR1 are chromosomally stable. (a) Flag-mBubR1 transgenic vector design. pCAGGS = promoter consisting of the CMV immediate enhancer and the chicken beta-actin promoter. (b) Western blots of tissue and MEF extracts from wildtype and Flag-BubR1 transgenic mice (strains T7 and T23). Tubulin was used as a loading control. (c) Both Flag-BubR1 transgenes correct the growth retardation and aging-associated phenotypes of BubR1 H/H mice. (d) High BubR1 expression does not induce aneuploidy. Chromosome counts were done on splenocytes from 5-month-old mice and passage (P) 5 MEFs (n = 3 samples per genotype). Fifty spreads were counted per sample (150 total). Values represent means ± SD. (e) High BubR1 levels do not induce chromosome missegregation. (f) The mitotic checkpoint is not hyperactive at supranormal BubR1 levels. Three independent MEF lines per genotype were used in e and f . Values represent means ± SD in f .

Article Snippet: Blots were probed with antibodies for P-H3 Ser10 (Millipore; 06-570, 1:500), BubR1 (1:1000) Flag (Origene; TA100011, 1:1000), β-actin (Sigma; A5441, 1:40000), EGFP (Cell Signaling; 2956, 1:1000), Aurora B (BD Biosciences; 611083, 1:1000), Cdc20 (1:1000), Plk1 (1:500) and Hras (1:5000) (Santa Cruz Biotechnology, Inc.; SC-8358, SC-17783, and SC-520, respectively), Bub3 (1:1000), Mad2 (1:1000), and Bub1 (1:1000).

Techniques: Transgenic Assay, Plasmid Preparation, Western Blot, Expressing