Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: Generation and characterization of an endogenous Aurora B-inducible model. (a) Schematic representation of the alleles used in this study for the inducible overexpression of Aurora B (see Materials and Methods for details). In the Aurkblox-tet allele, the Aurora B endogenous promoter was replaced by Tet-P, a minimal CMV promoter that contains seven in tandem repeats of tetO sequences recognized by the transactivator regulatory proteins rtTA. The activity of Cre recombinase resulted in the removal of the puromycin (puro) resistance cassette and the generation of the Aurkbtet allele. HR, homologous recombination; TK, thymidine kinase. (b) Southern blot analysis of ES cell clones after homologous recombination, indicating the presence of the recombinant Aurkblox-tet allele in one of the clones. The location of the probe is indicated in panel a. (c) Quantitative RT-PCR data from Aurkb+/+, Aurkb+/lox-tet, and Aurkb+/tet MEFs, carrying the Rosa26M2rtTA/M2rtTA allele, in the absence or presence of doxycycline (Doxy) for 48 h. GAPDH was used as a control of expression. Data are means ± standard deviations (n = 3 separate clones). *, P < 0.05; **, P < 0.01 (Student's t test). AU, arbitrary units. (d) Immunodetection of Aurora B in Aurkb+/+; Rosa26M2rtTA/M2rtTA, Aurkb+/lox-tet; Rosa26M2rtTA/M2rtTA and Aurkb+/tet; Rosa26M2rtTA/M2rtTA primary mouse embryonic fibroblasts (MEFs) stimulated with doxycycline. α-Tubulin (α-tub) was used as a loading control. (e) Aurora B levels in serum-starved Aurkb+/lox-tet; Rosa26M2rtTA/M2rtTA MEFs treated or not treated with doxycycline for 48 h. α-Tubulin was used as a loading control. (f) Aurora B expression is significantly induced in Aurkb+/lox-tet resting (0 h) or concanavalin A plus lipopolysaccharide (ConA+LPS)-induced (48 and 96 h) splenocytes. PS, Ponceau S. (g) EdU incorporation and DNA content profiles in Aurkb+/lox-tet splenocytes before or after treatment with concanavalin A plus lipopolysaccharide (ConA+LPS). Data are representative of two separate experiments.

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: Over Expression, Activity Assay, Homologous Recombination, Southern Blot, Clone Assay, Recombinant, Quantitative RT-PCR, Expressing, Immunodetection

Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: Localization and activity of overexpressed Aurora B. (a) Detection of Aurora B (green in the merged image) by immunofluorescence in the absence or presence of doxycycline. Data are representative of 50 mitotic cells analyzed per condition. Scale bars, 10 μm. (b) Protein levels of the CPC components in Aurora B-overexpressing and control MEFs. α-Tubulin was used as a loading control. (c) Phosphorylation of Hec1 (phospho-S55; pHec1) in prometaphase Aurora B-overexpressing cells. rel, relative. (d) Phosphorylation of histone H3 (pH3) in prophase or prometaphase/metaphase Aurora B-overexpressing cells. In panels a, c, and d, DAPI (blue) was used to stain DNA, and anticentromere antigen (ACA; red) was used as a control of centromeric localization. In panels c and d, horizontal bars indicate means (n = 10 to 12 mitotic cells per condition). ns, not significant; *, P = 0.05 (Student's t test).

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: Activity Assay, Immunofluorescence, Staining

Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: Overexpression of Aurora B results in chromosome segregation defects. (a) Proliferation of early-passage primary MEFs with or without the addition of doxycycline. Aurkb+/+ and Aurkb+/tet cells (always in the presence of the Rosa26M2rtTA allele) were treated with doxycycline or left untreated for 5 days. Data are representative of two different clones per condition. (b) Percentage and distribution of mitotic cells indicating the percentage of prometaphase (PM), metaphase (M), anaphase (A), or telophase (T) cells 48 h after the addition of doxycycline (n = 400 cells per treatment). (c) Percentage of abnormal mitotic figures (left panel) or abnormal interphasic cells (right panel) (bi-, multi-, and micronucleated cells; as shown in panel d) in Aurkb-overexpressing cells (n = 50 cells per treatment). (d) Percentage of mitotic defects (lagging or misaligned chromosomes and aberrant spindles), cytokinesis figures, and nuclear alterations in interphase in Aurkb+/tet MEFS in the absence or presence of doxycycline. Representative pictures are shown in the upper parts of the graphs. DAPI was used to stain DNA. Scale bars, 5 μm. n = 400 cells per condition. (e) Cytogenetic analysis of immortalized MEFs after exposure to 5 h of colcemid. The absolute numbers of chromosomes per metaphase were grouped in categories at early (passage 2) or late (passage 30) immortal passages (iP2 or iP30, respectively). Data are means ± standard deviations. n, number of metaphases analyzed per genotype. (f) Representative images of chromosome spreads from the indicated cultures. n, number of chromosomes per metaphase. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test).

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: Over Expression, Clone Assay, Staining

Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: Overexpression of Aurora B results in increased duration of mitosis accompanied by enhanced kinetochore loading of BubR1. (a) Schematic representation of the protocol used for time-lapse microscopy. Confluent cultures of immortalized Aurkb+/tet; Rosa26M2rtTA/M2rtTA MEFs were infected with adenoviruses (Adeno) expressing GFP-tagged histone H2B (H2B-GFP) following serum deprivation for 48 h. These cells were stimulated with serum and treated or not treated with doxycycline for 18 h. Then, the cells were left untreated or exposed to different drugs; next, they were monitored by using time-lapse microscopy during an additional 48 h. (b) Representative images of the indicated cultures during mitosis. The numbers of cells analyzed is indicated in each frame. Scale bars, 10 μm. H2B-GFP is in green. (c) Duration of mitosis in immortal untreated Aurkb+/tet (n = 74 cells) or doxycycline-induced Aurkb+/tet (n = 82 cells) MEFs showing an increase in the duration of mitosis when cells overexpress Aurora B. No differences were found when cells were exposed to 3.5 μM nocodazole or 1 μM paclitaxel. (d) The delay in mitotic exit upon Aurora B overexpression was rescued by the Aurora B inhibitor ZM447439 (n = 49 to 60 cells per condition). (e) The delay in mitotic exit upon Aurora B overexpression was rescued by the Mps1 inhibitor reversine (n = 65 to 100 cells per condition). Ratio between BubR1 (f) and Mad2 (g) to anticentromere antigen (ACA) signal in Aurkb+/tet MEFs treated or not treated with doxycycline (n = at least 70 centromere signals per group). Representative images of BubR1 and Mad2 (green) staining at kinetochores in untreated or doxycycline-treated Aurkb+/tet MEFs are also shown. ACA is shown in red, and DNA (DAPI) is in blue. Three different clones from each genotype/condition were analyzed. RU, relative units. Scale bars, 10 μm. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test). Horizontal bars indicate means.

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: Over Expression, Time-lapse Microscopy, Infection, Expressing, Staining, Clone Assay

Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: Tumor susceptibility in Aurora B-overexpressing mice. (a) Spontaneous tumor incidence (one tumor or more than one tumor per mouse) and type of neoplasias found in control (n = 7) or Aurora B-overexpressing (n = 12 Aurkblox-tet and 15 Aurkbtet) mice. ns, not significant; *, P < 0.05; ***, P < 0.001 (chi-square test). (b) Hematoxylin and eosin staining of the indicated tumors found in Aurkb+/tet; Rosa26M2rtTA/M2rtTA mice. Frames i and ii, spleen lymphoma; frame iii, histiocytic sarcoma; frame iv, lung adenoma; frame v, liver hemangioma; frame vi, lipogranuloma. Scale bars, 100 μm. Frame ii is an enlargement of the boxed area in frame i. (c) Representative pictures of CD3 and Pax5 staining in tumor samples from the spleen, liver and pancreas of Aurkb+/tet; Rosa26M2rtTA/M2rtTA mice. Scale bars, 50 μm. (d) Representative images of lymphoma metastasis in the lung (i), liver (ii), kidney (iii), and bone marrow (iv) in Aurkb+/tet; Rosa26M2rtTA/+ mice. Scale bars, 200 μm.

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: Staining

Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: Overexpression of Aurora B impairs the DNA damage response. (a) Schematic representation of the DNA damage assay performed in Aurkb+/tet; Rosa26M2rtTA/M2rtTA and Aurkb+/+; Rosa26M2rtTA/M2rtTA MEFs. Cells were treated with doxycycline (Doxy) for 24 h and doxorubicin (Adriamycin, Adr) was added for 2 h in order to induce p53 signaling. The protein levels of the indicated proteins were assessed before or at 10 h post-damage induction, and the fold change versus that in nondamaged cells is shown in the right histograms. (b) mRNA levels of Aurkb and Cdkn1a (encoding p21Cip1) transcripts in doxycycline-treated Aurkb+/tet; Rosa26M2rtTA/M2rtTA and Aurkb+/+; Rosa26M2rtTA/M2rtTA MEFs at the indicated times (0, 6, or 10 h) after treatment with doxorubicin (Adriamycin, Adr). GAPDH transcripts were used for normalization. Data are representative of three experiments. (c) Levels of the indicated antigens in the absence (−) or presence (10 h) of doxorubicin (Adriamycin, Adr) treatment and/or doxycycline (Doxy). The histogram shows the means ± standard deviations (n = 4 independent assays) of the change of these protein levels upon doxorubicin treatment in control or Aurkb+/tet cells treated with doxycycline. (d) To induce DNA damage in vivo, 6- to 8-week-old Aurkb+/+; Rosa26M2rtTA/M2rtTA (+/+ mice) and Aurkb+/tet; Rosa26M2rtTA/M2rtTA (+/tet) mice were treated for 15 days with doxycycline and irradiated (IR; 8 Gy of gamma irradiation). Cells positive for the corresponding antigens are indicated by arrows. The levels of the indicated proteins were tested by immunohistochemistry in the spleen 24 h later. γH2AX, phosphorylated H2AX; AC3, active caspase 3. Scale bars, 50 μm. Data in the histograms indicate means ± standard deviations. At least 15,000 cells were counted per genotype and condition (n = 4 irradiated and 2 nonirradiated mice). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test).

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: Over Expression, In Vivo, Irradiation, Immunohistochemistry

Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: In vivo overexpression of Aurora B induces aneuploidy. (a) Expression of Aurora B transcripts in the indicated tissues from Rosa26M2rtTA/M2rtTA mice harboring the Aurkblox-tet and Aurkbtet alleles after treatment with doxycycline for 2 weeks. mRNA levels were normalized to the expression of Gapdh mRNA. Data are representative of three assays (2 mice per genotype). ns, not significant; *, P < 0.05; **, P < 0.01. (b) Immunodetection of Aurora B in MEFs of the indicated tissues from Rosa26M2rtTA/M2rtTA mice harboring the Aurkblox-tet and Aurkbtet alleles. α-Tubulin (α-tub) and Ponceau S (PS) were used as loading controls. wt, wild type. (c) Immunohistochemical detection of Aurora B for the indicated tissues and genotypes (n = 3 mice per condition). Scale bars, 50 μm. (d) Levels of aneuploid cells in Aurkb+/+; Rosa26M2rtTA/M2rtTA and Aurkb+/tet; Rosa26M2rtTA/M2rtTA mice after 4 or 20 months in the presence of doxycycline. Aneuploidy (deviation from the mode) was scored using FISH for two different chromosomes in lymphocytes from 4- and 20-month-old animals (at least n = 100 cells from each of four animals per genotype and time point). Each column represents one animal of the indicated genotype. **, P < 0.01; ***, P < 0.001 (Student's t test). (e) Representative images of interphasic FISH analysis using probes for chromosomes 8 (red) and 11 (green). At least 100 cells from each of four animals per genotype and time point were analyzed. Scale bars, 20 μm.

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: In Vivo, Over Expression, Expressing, Immunodetection, Immunohistochemical staining

Journal: Molecular and Cellular Biology

Article Title: Aurora B Overexpression Causes Aneuploidy and p21 Cip1 Repression during Tumor Development

doi: 10.1128/MCB.01286-14

Figure Lengend Snippet: Aneuploidy and reduced p21Cip1 levels in Aurora B-overexpressing tumors. (a) Percentage of aneuploid cells in nontumoral (NT) spleens or spleens with initial (small, nonmetastatic) or fully developed tumors (T) from the indicated genotypes. FISH was performed for chromosomes 8 (data not shown) and 11 (red in the image) on spleen sections from 80-week-old mice. Each column represents one animal of the indicated genotype. Representative images are shown at the bottom. DNA is shown in blue, and FISH probes to chromosome 8 and 11 are in green and red, respectively. Scale bar, 20 μm. At least 100 cells were counted per condition. (b) Percent of near-tetraploid cells and aneuploid cells (excluding those scored as near-tetraploid) determined by chromosome FISH hybridization in nontumoral (NT) or tumoral (either initial, Init-T or fully developed, T, tumors) spleen samples from Aurkb+/+; Rosa26M2rtTA/M2rtTA and Aurkb+/tet; Rosa26M2rtTA/M2rtTA mice. Each column represents one animal of the indicated genotype. At least 100 cells were counted per condition. (c) Immunodetection of p53 in nontumoral (NT) or tumoral (either initial or fully developed) spleen samples from Aurkb+/+; Rosa26M2rtTA/M2rtTA and Aurkb+/tet; Rosa26M2rtTA/M2rtTA mice. Scale bar, 80 μm. The quantification of the percentage of p53-positive cells (means ± standard deviations; n >10,000 cells per genotype and condition) is shown in the histogram. (d) p21Cip1 levels in nontumoral (NT) and tumoral (T) spleen samples from Aurkb+/+ mice in comparison to levels in initial and fully developed tumors from Aurkb+/tet mice. Scale bar, 80 μm. The percentage of p21Cip1-positive cells (means ± standard deviations; n > 11,000 cells per genotype and condition) is shown in the histogram. Significance in panels a to d is indicated as follows: *, P < 0.05; ***, P < 0.001 (Student's t test). (e) Inverse correlation between AURKB and CDKN1A mRNA levels in 207 samples of human B-cell acute lymphoblastic leukemia (B-ALL). Expression data obtained from Kang et al. and Harvey et al. (31, 32). Pearson correlation (R2) and P values are indicated.

Article Snippet: Additional immunohistochemical examination of the tissues and pathologies was performed using specific antibodies against the following antigens: Aurora B (ab2254; Abcam), Ki67 (0003110QD; Master Diagnostica), active caspase 3 (AF835; R&D Systems), CD3e (M-20, sc-1127; Santa Cruz Biotechnology), phospho-p53 (Ser15; Cell Signaling), p21 Cip1 (sc-397-G; Santa Cruz Biotechnology), Pax-5 (C-20, sc-1974; Santa Cruz Biotechnology), or γ-H2AX (05-636; Millipore).

Techniques: Hybridization, Immunodetection, Expressing

Journal:

Article Title: Aurora-B Regulates RNA Methyltransferase NSUN2

doi: 10.1091/mbc.E06-11-1021

Figure Lengend Snippet: Antibody against Histone H3 phosphorylated at Ser10 binds to unknown 100-kDa protein. (A) The 100-kDa protein (arrowhead) is phosphorylated in cells treated for 18 h with nocodazole, and its phosphorylation is inhibited by the expression of Aurora-B-K/R but not Aurora-A-K/R. (B) The 100-kDa protein is phosphorylated during mitosis in synchronized cells. (C) Coomassie brilliant blue staining of the 100-kDa protein immunoprecipitated by αH3-P. Synchronized HeLa cells were collected at the indicated times, and the immunoprecipitated samples and the supernatant were separated by SDS-PAGE. (D) Immunoblot of the 100-kDa protein immunoprecipitated by αH3-P. Samples shown in C were analyzed by immunoblotting with αH3-P. In C and D, asterisks indicate the immunoprecipitated 100-kDa protein. IB, immunoblot; IP, immunoprecipitation; H3, Histone H3; WT, wild-type; K/R, kinase negative form; exp, exponentially growing; Noc, nocodazole treatment.

Article Snippet: Other antibodies used in the experiments included the following: polyclonal rabbit anti-Cdc25A ( Jinno et al. , 1994 ) and anti-Human Aurora-C antibodies (38-9400; Zymed Laboratories, South San Francisco, CA); and monoclonal antibodies against FLAG (M5; Sigma-Aldrich, St. Louis, MO), Xpress (Invitrogen), α-tubulin (CLT-9002; Cedarlane Laboratories, Hornby, Ontario, Canada), enhanced green fluorescent protein (EGFP; JL-8; BD Biosciences), Aurora-A (clone 4; BD Biosciences), Aurora-B ( Kanda et al. , 2005 ), fibrillarin (38F3; Abcam, Cambridge, United Kingdom), NPM1 (B0556; Sigma-Aldrich), and nucleolin (sc-8031; Santa Cruz Biotechnology).

Techniques: Expressing, Staining, Immunoprecipitation, SDS Page, Western Blot

Journal:

Article Title: Aurora-B Regulates RNA Methyltransferase NSUN2

doi: 10.1091/mbc.E06-11-1021

Figure Lengend Snippet: NSUN2 is phosphorylated during mitosis on Ser139 by Aurora-B. (A) NSUN2 is constitutively expressed and possibly phosphorylated during mitosis. The samples were collected at the indicated times in synchronized HeLa cells. The top, middle, and bottom panels show immunoblots (IB) using αH3-P, αNSUN2, and anti-α-tubulin antibody, respectively. Arrowhead indicates the phosphorylated 100-kDa protein, presumably phosphorylated by NSUN2. (B) NSUN2 is expressed even in the quiescent state. NHDF cells were cultured under exponential (exp) growth conditions or in 0.2% fetal bovine serum for 48 h (G0). (C) NSUN2 is phosphorylated during mitosis but not during interphase in synchronized HeLa cells. (D) NSUN is phosphorylated by Aurora-B in vitro. (E) NSUN2 phosphorylated in vitro by Aurora-B is recognized by αH3-P. The samples shown in D were analyzed by immunoblotting using αH3-P. (F) Aurora inhibitors Hesperadin and ZM447439 repress NSUN2 and Histone H3 phosphorylation during mitosis in synchronized HeLa cells. Eight hours after release, the cells were treated with inhibitors for 2 h. The control lysate was collected immediately after release. (G) Aurora-B-K/R and Aurora-C-K/R repress the phosphorylation of NSUN2 during mitosis in ΔCyclin B1-arrested cells. HeLa cells were transfected with the indicated pair of plasmids, and after 18 h, cells were lysed and analyzed by immunoblotting by using αH3-P, αNSUN2, anti-EGFP antibody, or anti-FLAG antibody. (H) Aurora-B-shRNA represses the phosphorylation of NSUN2 during mitosis in ΔCyclin B1-arrested cells. HeLa cells were transfected with the indicated pair of plasmids, and after treatment for 48 h with puromycin, cells were lysed and analyzed by immunoblotting αH3-P, αNSUN2, anti-EGFP antibody, or anti-Aurora-A, -B, or -C antibodies. H3, Histone H3; H3-P, phosphorylated Histone H3; NSUN2-P, phosphorylated NSUN2; Aurora-B-P, phosphorylated Aurora-B; IB, immunoblot; IP, immunoprecipitation.

Article Snippet: Other antibodies used in the experiments included the following: polyclonal rabbit anti-Cdc25A ( Jinno et al. , 1994 ) and anti-Human Aurora-C antibodies (38-9400; Zymed Laboratories, South San Francisco, CA); and monoclonal antibodies against FLAG (M5; Sigma-Aldrich, St. Louis, MO), Xpress (Invitrogen), α-tubulin (CLT-9002; Cedarlane Laboratories, Hornby, Ontario, Canada), enhanced green fluorescent protein (EGFP; JL-8; BD Biosciences), Aurora-A (clone 4; BD Biosciences), Aurora-B ( Kanda et al. , 2005 ), fibrillarin (38F3; Abcam, Cambridge, United Kingdom), NPM1 (B0556; Sigma-Aldrich), and nucleolin (sc-8031; Santa Cruz Biotechnology).

Techniques: Western Blot, Cell Culture, In Vitro, Transfection, shRNA, Immunoprecipitation

Journal: eLife

Article Title: Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart

doi: 10.7554/eLife.50163

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were anti-AuroraB kinase (BD Transduction laboratories #611082, 1:200), anti-Ki67 (Cell Marque #275R, 1:200), anti-MCT4 (Santa Cruz #SC50329, 1:200), anti-PCNA (Dako #M0879, 1:800), anti-GFP (aves #GFP-1010, 1:1000), and anti-Mef2c (Santa Cruz #SC313, Biorbyt #orb256682 both 1:1000).

Techniques: Transgenic Assay, Inhibition, Transduction, SYBR Green Assay, Software

Journal: Scientific Reports

Article Title: Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

doi: 10.1038/srep40714

Figure Lengend Snippet: ( a ) Panel of bladder cancer cells shows different levels of AURKA expression in UC5, UC6, UC7, UC 9, UC10 and UC11 cell lines. Note low level of AURKA expression in cultured normal urothelial (NHU) cells. Full length blots are shown in . ( b ) Using a lentiviral shRNA construct AURKA was silenced by ~75% in UC7 and UC11 cells. Full length blots are shown in . ( c ) Heatmap of most significantly up- and down-regulated genes after silencing of AURKA in UC11 cell line. ( d ) AURKA was silenced using a lentiviral vector. Upon silencing, NNMT is upregulated by 3–4 fold in UC7 and UC11 cell lines. In contrast, overexpression of AURKA in UC5 cells resulted with downregulation of NNMT. Full length blots are shown in . ( e ) Upon silencing of AURKA cell invasion is decreased by ~3 fold in both UC7 and UC11 cells in a matrigel invasion assay (*P < 0.01). ( f ) A luciferase promoter analysis of NNMT reveals that NNMT’s upregulation is transcriptionally regulated in both cell lines upon silencing of AURKA (*P < 0.05). ( g ) AURKA KO UC7 and UC11 cells were treated with AURKA rescue vectors and western blot was performed to verify the rescue of AURKA and NNMT expression. Full length blots are shown in . ( h ) Quantitation of cell invasion through matrigel coated invasion chambers in AURKA silenced and rescued UC7 and UC11 cell lines (*P < 0.05). ( i ) Luciferase based promoter analysis of the NNMT promoter indicated that when AURKA is rescued, luciferase expression is returned to lower than normal levels in both UC7 and UC11cell lines (*P < 0.05). ( j ) Overexpression of AURKA in UC5 cells which resulted in downregulation of NNMT (see Fig. 1d) increased cell invasion in matrigel coated chambers. ( k ) NNMT was silenced 80–90% in UC7 and UC11 cell lines using lentiviral shRNA directed against NNMT. A reduction of NNMT expression was observed by western blot. Full length blots are shown in . ( l ) An increase in cell invasion was observed in both cell lines in NNMT shRNA treated cells when compared to NT shRNA (*P < 0.05).

Article Snippet: Immunohistochemical staining for AURKA (Gene Tex GTX21287) and NNMT (Abcam, ab119758) was performed according to a standard immunohistochemical protocol.

Techniques: Expressing, Cell Culture, shRNA, Construct, Plasmid Preparation, Over Expression, Invasion Assay, Luciferase, Western Blot, Quantitation Assay

Journal: Scientific Reports

Article Title: Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

doi: 10.1038/srep40714

Figure Lengend Snippet: ( a ) An NNMT activity assay was performed on UC7 and UC11 cell lines. The activity of NNMT normalized to actin correlated with its expression levels (*P < 0.05, **P < 0.01). ( b ) Western blotting revealed that expression levels of PAX3 decrease upon silencing of AURKA. Full length blots are shown in . ( c ) Schematic presentation of the NNMT promoter. Two PAX3 binding sites are located within 500 bp upstream of the NNMT transcription start site. ( d ) Chromatin immunoprecipitation indicates that PAX3 binds to the NNMT promoter and represses its expression in both UC7 (top) and UC11 (bottom) cell lines. Full length gels are shown in . ( e ) Luciferase based promoter analysis of the NNMT promoter with mutations in either of the PAX3 binding sites leads to an increase in luciferase expression in the non-targeted (NT) cells to similar levels as the AURKA KO cells. Mutation at both sites together did not have an additive effect, indicating that both sites are necessary for suppression of NNMT transcription (*P < 0.05).

Article Snippet: Immunohistochemical staining for AURKA (Gene Tex GTX21287) and NNMT (Abcam, ab119758) was performed according to a standard immunohistochemical protocol.

Techniques: Activity Assay, Expressing, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Mutagenesis

Journal: Scientific Reports

Article Title: Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

doi: 10.1038/srep40714

Figure Lengend Snippet: ( a ) Western blots for MMP2 and MMP9 indicate an increase in both MMPs in NNMT KO cells and a decrease in both AURKA KO cells. Full length blots are shown in . ( b ) Zymogram indicates increased levels of pro- and active forms of MMP2 in both UC7 and UC11 cell lines in NNMT KO cells, while a decrease in both pro- and active forms was observed in AURKA KO cells. Full length zymograms are shown in . ( c ) MMP2 ELISA shows higher MMP2 levels in NNMT KO cells and lower levels in AURKA KO cells in both UC7 and UC11 cell lines (*P < 0.05, **P < 0.01).

Article Snippet: Immunohistochemical staining for AURKA (Gene Tex GTX21287) and NNMT (Abcam, ab119758) was performed according to a standard immunohistochemical protocol.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

doi: 10.1038/srep40714

Figure Lengend Snippet: ( a ) Expression levels of Aurora A and NNMT genes revealed by immunohistochemical staining in selected tumor samples from tissue microarray showing three paired patterns characteristic for three groups of invasive (T1 and higher) bladder cancer: GA (AURKA and NNMT with low expression), GB (AURKA and NNMT with high expression) and GC (AURKA with high expression and NNMT with low expression). ( b ) Kaplan-Meier analyses of disease-specific survivals in four groups of samples. Superficial (Ta) tumors were separated from invasive (T1 and higher). The invasive tumors were divided into three groups corresponding to samples with (1) low levels of AURKA expression (AURKA-L); (2) high levels of AURKA (AURKA-H), and high levels of NNMT (NNMT-H); and (3) high levels of AURKA (AURKA-H) and low levels of NNMT (NNMT-L). ( c ) Expression pattern of AURKA and AURKA signature genes in molecular subtypes of bladder cancer in the TCGA bladder cancer cohort comprising 128 high-grade muscle invasive bladder tumor samples which were classified into luminal, p53-like, and basal subtypes using the previously published algorithm. Subsets of samples with high levels of AURKA (AURKA-H) as well as subsets of samples showing overexpression of AURKA (AURKA-H) with downregulation of NNMT in molecular subtypes are shown. ( d ) Expression levels of AURKA and NNMT in luminal and basal subtypes of bladder cancer. ( e ) Proportions of cases with overexpression of AURKA (AURORA-H) and proportions of cases which show overexpression of AURKA (AURKA-H) with downregulation of NNMT (NNMT-L) in luminal and basal subtypes of bladder cancer. ( f ) Enrichment of AURKA downstream regulatory pathways in basal as compared to luminal subtypes of bladder cancer revealed by GSEA.

Article Snippet: Immunohistochemical staining for AURKA (Gene Tex GTX21287) and NNMT (Abcam, ab119758) was performed according to a standard immunohistochemical protocol.

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Over Expression

Journal: Scientific Reports

Article Title: Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

doi: 10.1038/srep40714

Figure Lengend Snippet: ( a ) Dual-fluorescence FISH test with probes for AURKA (red) and the chromosome 20 α-satellite DNA (green) performed on 2 pairs of tissue and urine from the same patient with LGTCC and HGTCC respectively. Nuclei were counterstained with DAPI (blue). ( b ) Quantitative FISH analysis of AURKA gene copy numbers in tissue and voided urine specimens from 20 patients. The percentage of abnormal cells with more than 2 copies of AURKA in the individual patients is shown. ( c ) Mean percentage of cells with 3–4 copies and more than 4 copies of AURKA in matching tissue and urine samples from patients with LGTCC and HGTCC. ( d ) Average proportion of cells in voided urine showing 3–4 or more than 4 copies of AURKA in LGTCC and HGTCC detected in voided urine. ( e ) Receiver operating characteristic (ROC) curve for the set consisting of 232 urine samples from patients with bladder cancer and 255 urine samples from control subjects (126 healthy controls and 129 individuals with benign non-neoplastic disorders of the urinary tract). The AURKA FISH test for the detection of bladder cancer showed an area under the receiver operating characteristic curve (AUC) of 0.895 (95% confidence interval [CI] = 0.984 to 1.000). ( f ) AURKA gene FISH score in voided urine by histological grade of TCC stratified into low and high groups. Horizontal bars designate the mean AURKA gene score for TCCs of low and high histological grade. P value was calculated from two-sided Mann–Whitney t test. ( g ) Kaplan-Meier analyses of overall survival in low-grade (G1-2) and high grade (G3) tumors according to AURKA score. ( h ) Kaplan-Meier analyses of overall survival in superficial (Ta) and invasive (T1 and >) tumors according to AURKA score. ( i ) Comparison of AURKA FISH test results with cytologic analyses of voided urine in 144 samples from patients with bladder cancer.

Article Snippet: Immunohistochemical staining for AURKA (Gene Tex GTX21287) and NNMT (Abcam, ab119758) was performed according to a standard immunohistochemical protocol.

Techniques: Fluorescence, Control, MANN-WHITNEY, Comparison