β-catenin Search Results


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  • 99
    Cell Signaling Technology Inc β catenin
    Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). <t>β-catenin</t> immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.
    β Catenin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology β catenin
    Immunohistochemical analysis of <t>β-catenin</t> and c-Myc protein expression in the venous limb of the murine AVF 1 wk after the creation of the AVF. Immunohistochemical localization of β-catenin protein in control vein ( A ) and the venous segment
    β Catenin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 5488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson β catenin
    Immunofluorescence analysis of paraffin-embedded sections of APC mutant mouse intestines. ( a ) HE-stained section of the intestines of an 16-weeks old Apc Δ716 mouse. Polyps analyzed in this study are indicated by numbers. ( b ) Immunostaining of a formalin-fixed, paraffin-embedded Apc Δ716 mouse intestine tissue section for E-cadherin and <t>β-catenin</t> with nuclear DNA stained by Hoechst 33342 to visualize crypt cells (top) and tumour cells (bottom) is shown. Low magnification images are shown on the left, and magnified images of the boxed areas are shown in the four panels on the right. Note that β-catenin levels are upregulated but its preferential accumulation to the nucleus is not observed. (c) Upregulation of TPX2 in Apc Δ716 tumour tissues. Immunostaining of a paraffin-embedded section for TPX2 with nuclear DNA stained by Hoechst 33342 is shown. Crypt cells (top) and tumour cells (bottom) were observed. Scale bars: 50 μm (b, low mag, left), 10 μm (b, high magnification, right), and 5 μm (c).
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    Abcam β catenin
    Association of four epithelial-mesenchymal transition-a ssociated markers with overall survival. Kaplan-Meier overall survival curves stratified by the expression of (A) E-cadherin, (B) N-cadherin, (C) <t>β-catenin</t> and (D) ZEB2. P-values were calculated using the log-rank test. ZEB2, zinc finger E-box binding homeobox 2.
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    Cell Signaling Technology Inc anti β catenin
    Protein expressions of Wnt pathway molecules after downregulation of Gal-3 in ovarian cancer cell lines. Notes: Downregulating Gal-3 expression attenuates <t>β-catenin</t> expression in ovarian epithelial cancer cells SKOV3 and OVCAR3; c-myc and cyclin D1 which are both downstream proteins in Wnt pathways are downregulated. Abbreviations: Gal-3, galectin-3; NC, negative control; siRNA, small interfering RNA.
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    Millipore β catenin
    CK1ε depletion induces apoptosis and growth inhibition through activating <t>β-catenin.</t> ( A ) β-catenin signaling in CK1ε-deficient U87MG cells. Phosphorylated, active, and total β-catenin was assessed using immunoblotting. ACTB (β-actin) is the loading control. Band intensities were quantified using Image J. ( B ) β-catenin signaling in CK1ε-deficient A172 cells. ( C ) Luciferase reporter assay. The TOPFlash plasmid harbors TCF/LEF binding sites and responds to β-catenin activation. FOPFlash contains mutated TCF/LEF binding sites and does not respond to β-catenin activation. ( D ) Immunoblotting of cleaved caspase 3 (c-CASP3, an apoptosis marker) and LC3B (an autophagy marker). ( E ) Caspase 3/7 activity assay. ( F ) Knockdown of β-catenin. U87MG cells were transduced with viruses of NS shRNA or β-catenin shRNA. ( G ) Viability of U87MG cells treated with CK1ε shRNA and/or β-catenin shRNA. ( H ) Immunoblotting of c-CASP3 in U87MG cells upon depletion of CK1ε and/or β-catenin. Error bars represent standard deviations from three independent experiments. Full length blots were presented in supplemental materials. * P
    β Catenin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti β catenin
    Aspirin treatment increases expression of intracellular DKK-1 while reducing secreted DKK-1. ( A ) Immunoblotting for DKK-1 and LRP6 protein abundance in whole cell extracts and secreted DKK-1 protein abundance in media from HCT116 cells treated with 0.5 mmol/L aspirin, 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 and 24 hours. ( B ) Immunoblotting for DKK-1 and LRP6 protein abundance in whole cell extracts and secreted DKK-1 protein abundance in media from Colo205 cells treated with 0.5 mmol/L aspirin, 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 and 24 hours. ( C ) Immunoblotting of DKK-1, c-myc, and Snail protein abundance in cytoplasmic/nuclear extracts from HCT116 cells treated with 3 mmol/L aspirin for 24 hours. Immunoblotting data representative of 3 independent experiments. ( D ) DKK-1 transcript expression in organoids derived from human FAP normal colonic mucosa tissue and adenomatous tissue treated with 2 mmol/L aspirin for 4 hours. DKK-1 transcript levels are normalized to 18S transcripts, and data are expressed as fold-change compared with untreated control sample. DKK-1 transcript data represent 3 individual FAP patient samples. ( E ) DKK-1 transcript expression in fetal organoids treated with 2 mmol/L aspirin for 8 days. DKK-1 transcript levels are normalized to 18S transcripts, and data are expressed as fold-change compared with untreated control sample. ( F ) Average size of fetal organoids treated with 2 mmol/L aspirin for 8 days. Fetal organoid data represent 4 independent experiments. (G) Immunoblotting of DKK-1, <t>β-catenin,</t> E-cadherin, SOX-9, c-myc, and Snail protein abundance in untransfected HCT116 cells and HCT116 cells transfected with siRNA targeting DKK-1 or control siRNA. Cells were treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours. β-actin represents sample control. DKK-1 siRNA data representative of 3 independent experiments. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by Student t test. Asterisks denote P value (∗
    Anti β Catenin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 3197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher β catenin
    Periostin suppressed the osteogenic differentiation of suture-derived cells via inactivation of <t>Wnt/β-catenin</t> signaling. a The mRNA expression of Wnt-7b, Wnt-1, Wnt-3a, and β-catenin of suture-derived cells after treatment with different concentrations of recombinant mouse periostin. b Western blotting analysis of Wnt/β-catenin signaling markers. c Quantification of Western blotting results. *P
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    Becton Dickinson mouse anti β catenin
    IpaC Stabilizes the <t>β-Catenin-Cadherin</t> Interaction (A–C) Infection of Caco-2 cells with S. flexneri -producing WT IpaC or IpaC R362W. (A) Representative images. Five to ten images were analyzed per condition per experiment. Cyan, S. flexneri ; green, membrane-bound YFP; red, β-catenin; blue, DNA. Scale bar 20 βM. (B and C) Quantification of the percentage of protrusions that colocalized with β-catenin in either sub-confluent (B) or confluent (C) cells. Student’s t test. Data are mean ± SEM. (D) Subcellular localization of β-catenin in HeLa cells infected with S. flexneri -producing WT IpaC or IpaC R362W. (E–G) Yeast-based protein interaction assay comparing the efficiency of the interaction between β-catenin and E-cadherin in the presence of either WT IpaC or IpaC R362W. (E) Schematic showing prey is E-cadherin GFP, modifier is IpaC, and bait is β-catenin-μNS. (F) Representative images. Dotted lines are outlines of yeast. Scale bar, 10 μm. A total of 29–175 yeast were analyzed per condition per experiment. (G) Percentage of yeast displaying puncta, which indicates an interaction, from (F). Data are mean ± SEM. N.S., not significant. *p
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    Millipore anti β catenin
    Effects of Wnt modulation and Fluoxetine treatment on pain thresholds. (A,B) Effects of ablation of <t>β-catenin</t> in hippocampal neural progenitors on mechanical and thermal withdraw thresholds of SNI treated mice. (C,D) Effects of expressing stabilized β-catenin in hippocampal neural progenitors on mechanic and thermal withdraw thresholds of SNI treated mice. (E,F) Effects of Fluoxetine treatment on mechanic and thermal withdraw thresholds of sham injured and SNI treated mice. S, saline. F, Fluoxetine. Notice the no change of pain thresholds in mice with β-catenin manipulation or Fluoxetine treatment. Values represent mean ± SE. One-way ANOVA analysis with Dunnett’s post hoc test was performed in (A–F) . * P
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    Cell Signaling Technology Inc rabbit anti β catenin
    The increased mRNA and protein levels of <t>β-catenin</t> were partially reversed by addition of a Wnt/β-catenin signaling inhibitor (DKK1). ( a ) The β-catenin mRNA levels in control, morroniside and morronside +DKK1 groups were determined by RT-PCR after treatment for 24 h. ( b ) Total β-catenin protein levels in control, morroniside and morronside +DKK1 groups were determined by western blotting. The grouping of blots was cropped from the same gel. ( c ) Quantitative analysis of β-catenin protein levels in control, morroniside and morroniside +DKK1 groups. a.u., arbitrary units.
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    Millipore anti beta catenin antibody
    The increased mRNA and protein levels of <t>β-catenin</t> were partially reversed by addition of a Wnt/β-catenin signaling inhibitor (DKK1). ( a ) The β-catenin mRNA levels in control, morroniside and morronside +DKK1 groups were determined by RT-PCR after treatment for 24 h. ( b ) Total β-catenin protein levels in control, morroniside and morronside +DKK1 groups were determined by western blotting. The grouping of blots was cropped from the same gel. ( c ) Quantitative analysis of β-catenin protein levels in control, morroniside and morroniside +DKK1 groups. a.u., arbitrary units.
    Anti Beta Catenin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho β catenin
    The increased mRNA and protein levels of <t>β-catenin</t> were partially reversed by addition of a Wnt/β-catenin signaling inhibitor (DKK1). ( a ) The β-catenin mRNA levels in control, morroniside and morronside +DKK1 groups were determined by RT-PCR after treatment for 24 h. ( b ) Total β-catenin protein levels in control, morroniside and morronside +DKK1 groups were determined by western blotting. The grouping of blots was cropped from the same gel. ( c ) Quantitative analysis of β-catenin protein levels in control, morroniside and morroniside +DKK1 groups. a.u., arbitrary units.
    Phospho β Catenin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti β catenin antibody
    Gene expression during neural crest induction in the zebrafish embryo and upon Wnt stimulation in SKMEL28 melanoma cells, and clinical significance. a Heatmaps of real-time qPCR gene expression analysis of the melanoma cell lines SKMEL19, 451 LU, BLM and A375 for AXIN2. TYR, MITF and the four genes INHBA, CYR61, ANGTPL4 and FABP7. Log2 transformed x-fold expression values were used for color-coding. Yellow: upregulated gene expression; blue: downregulated gene expression at 24 h treatment of the melanoma cells; black: not detectable b Western blot analysis to detect protein levels of <t>beta-catenin,</t> PTEN, phospo-Ser473 (AKT) and AKT in the melanoma cell lines A375, SKMEL19, BLM and 451 LU after treatment with 3T3-CM, 3T3-Wnt3a, or 0.5 μM PKF115–584 for 24 h. Beta-actin was used as loading control. Small-hairpin knockdown cells served as samples to investigate the dependence on beta-catenin. c Luciferase reporter assay (Super8xTOPFlash) indicates a 3-fold activation of the canonical <t>Wnt−/β-catenin</t> signaling pathway after stimulation of SKMEL28 cells with Wnt3a-conditioned medium (3T3-Wnt3a). PKF115–584 treatment (0.5 μM for 12 h) significantly inhibited reporter activity. **: p
    Anti β Catenin Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 641 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibody against β catenin
    Compared to oral gavage, intraperitoneal 4-AAQB significantly and more efficiently suppresses GBM stem cell-induced tumor growth, in vivo. ( A ) Tumor size vs time curve show the inhibitory effect of 4-AAQB on U87MG tumor growth either via p.o or i.p route, as compared to the control group. ( B ) Photographs of tumor samples harvested from the in vivo studies. ( C ) The differential effects of oral and intraperitoneal administration of 4-AAQB on the expression and localization of TCF-1 and <t>β-catenin</t> in xenograft-derived GBM primary culture. * p
    Antibody Against β Catenin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p β catenin
    Compared to oral gavage, intraperitoneal 4-AAQB significantly and more efficiently suppresses GBM stem cell-induced tumor growth, in vivo. ( A ) Tumor size vs time curve show the inhibitory effect of 4-AAQB on U87MG tumor growth either via p.o or i.p route, as compared to the control group. ( B ) Photographs of tumor samples harvested from the in vivo studies. ( C ) The differential effects of oral and intraperitoneal administration of 4-AAQB on the expression and localization of TCF-1 and <t>β-catenin</t> in xenograft-derived GBM primary culture. * p
    P β Catenin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 602 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti β catenin
    Inhibition of <t>Wnt/β-catenin</t> signaling impairs ELFEF-induced increases of SVZ neurogenesis and olfactory memory. ( a – c ) Bar graphs showing the number of BrdU + ( a ) and BrdU + /Nestin + ( b ) cells in the SVZ, and BrdU + /DCX + cells ( c ) in the RMS of control and ELFEF-exposed mice perfused with either vehicle (CTRL) or Dkk-1. ( d ) In the odor discrimination test, ELFEF-exposed mice injected with vehicle (ELFEF) showed a higher discrimination ability when compared to ELFEF-exposed mice injected with Dkk-1 (Dkk-1-ELFEF). ( e ) Injection of Dkk-1 into the SVZ abolished the enhancement of olfactory memory induced by ELFEF. ( f ) In the long-term olfactory memory tests, the preference index of Dkk-1-ELFEF mice was significantly less when compared to ELFEF mice. Values are expressed as means ± SEM. (n = 7–8 mice per group). n.s. = not significant, *p
    Rabbit Anti β Catenin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 541 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti beta catenin antibody
    Inhibition of <t>Wnt/β-catenin</t> signaling impairs ELFEF-induced increases of SVZ neurogenesis and olfactory memory. ( a – c ) Bar graphs showing the number of BrdU + ( a ) and BrdU + /Nestin + ( b ) cells in the SVZ, and BrdU + /DCX + cells ( c ) in the RMS of control and ELFEF-exposed mice perfused with either vehicle (CTRL) or Dkk-1. ( d ) In the odor discrimination test, ELFEF-exposed mice injected with vehicle (ELFEF) showed a higher discrimination ability when compared to ELFEF-exposed mice injected with Dkk-1 (Dkk-1-ELFEF). ( e ) Injection of Dkk-1 into the SVZ abolished the enhancement of olfactory memory induced by ELFEF. ( f ) In the long-term olfactory memory tests, the preference index of Dkk-1-ELFEF mice was significantly less when compared to ELFEF mice. Values are expressed as means ± SEM. (n = 7–8 mice per group). n.s. = not significant, *p
    Anti Beta Catenin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti active β catenin
    Deletion of <t>β-catenin</t> suppresses growth of menin-null PNETs and increases survival time. ( a ) Immunofluorescence staining of menin, β-catenin and insulin on pancreatic sections from 8-week-old mouse models. Scale bars, 50 μm. ( b ) The incidence of tumorigenesis in mouse models ( n =18–31). * P
    Anti Active β Catenin, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    β catenin cadherin associated protein beta 88 kDa is a dual function protein regulating the coordination of cell cell adhesion and gene transcription via the Wnt signaling pathway Mutations and
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    Ultra LEAF Purified anti β Catenin 1 CTNNB1 12F7 Isotype Mouse IgG1 κ Reactivity Human Mouse Rat Apps WB IF IHC Size 100 µg
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    Image Search Results


    Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). β-catenin immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.

    Journal: Journal of Toxicologic Pathology

    Article Title: Spontaneous pulmonary co-metastasis of hepatoblastoma arising within a hepatocellular carcinoma in an aged C57BL/6J mouse

    doi: 10.1293/tox.2017-0067

    Figure Lengend Snippet: Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). β-catenin immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm.

    Article Snippet: Endogenous peroxidase was blocked by incubating sections in 3% H2 O2 for 15 min. For antigen retrieval, sections were immersed in citrate buffer (pH 6.0), heated, and cooled at room temperature. β-catenin (rabbit monoclonal, clone 6B3, dilution 1:150, Cell Signaling) specific antibody was applied for 1.5 hours at room temperature.

    Techniques: Immunohistochemistry, Staining

    Immunohistochemical analysis of β-catenin and c-Myc protein expression in the venous limb of the murine AVF 1 wk after the creation of the AVF. Immunohistochemical localization of β-catenin protein in control vein ( A ) and the venous segment

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: ?-Catenin is markedly induced in a murine model of an arteriovenous fistula: the effect of metalloproteinase inhibition

    doi: 10.1152/ajprenal.00488.2010

    Figure Lengend Snippet: Immunohistochemical analysis of β-catenin and c-Myc protein expression in the venous limb of the murine AVF 1 wk after the creation of the AVF. Immunohistochemical localization of β-catenin protein in control vein ( A ) and the venous segment

    Article Snippet: Primary rabbit polyclonal antibodies to c-Myc and β-catenin (catalog nos. sc-764 and sc-1496R, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) were applied for 60 min at room temperature.

    Techniques: Immunohistochemistry, Expressing

    The effect of doxycycline on N-cadherin, β-catenin, and c-Myc protein expression in the venous limb of the murine AVF 1 wk after the creation of the AVF. Protein extracted from the venous segment of the AVF in mice with or without doxycycline

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: ?-Catenin is markedly induced in a murine model of an arteriovenous fistula: the effect of metalloproteinase inhibition

    doi: 10.1152/ajprenal.00488.2010

    Figure Lengend Snippet: The effect of doxycycline on N-cadherin, β-catenin, and c-Myc protein expression in the venous limb of the murine AVF 1 wk after the creation of the AVF. Protein extracted from the venous segment of the AVF in mice with or without doxycycline

    Article Snippet: Primary rabbit polyclonal antibodies to c-Myc and β-catenin (catalog nos. sc-764 and sc-1496R, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) were applied for 60 min at room temperature.

    Techniques: Expressing, Mouse Assay

    Western analysis of β-catenin protein expression in the venous limb of the murine AVF 1 wk after the creation of the AVF. Protein extracted from control and AVF veins was immunoblotted for β-catenin. Equivalency of protein loading was

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: ?-Catenin is markedly induced in a murine model of an arteriovenous fistula: the effect of metalloproteinase inhibition

    doi: 10.1152/ajprenal.00488.2010

    Figure Lengend Snippet: Western analysis of β-catenin protein expression in the venous limb of the murine AVF 1 wk after the creation of the AVF. Protein extracted from control and AVF veins was immunoblotted for β-catenin. Equivalency of protein loading was

    Article Snippet: Primary rabbit polyclonal antibodies to c-Myc and β-catenin (catalog nos. sc-764 and sc-1496R, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) were applied for 60 min at room temperature.

    Techniques: Western Blot, Expressing

    Immunofluorescence analysis of paraffin-embedded sections of APC mutant mouse intestines. ( a ) HE-stained section of the intestines of an 16-weeks old Apc Δ716 mouse. Polyps analyzed in this study are indicated by numbers. ( b ) Immunostaining of a formalin-fixed, paraffin-embedded Apc Δ716 mouse intestine tissue section for E-cadherin and β-catenin with nuclear DNA stained by Hoechst 33342 to visualize crypt cells (top) and tumour cells (bottom) is shown. Low magnification images are shown on the left, and magnified images of the boxed areas are shown in the four panels on the right. Note that β-catenin levels are upregulated but its preferential accumulation to the nucleus is not observed. (c) Upregulation of TPX2 in Apc Δ716 tumour tissues. Immunostaining of a paraffin-embedded section for TPX2 with nuclear DNA stained by Hoechst 33342 is shown. Crypt cells (top) and tumour cells (bottom) were observed. Scale bars: 50 μm (b, low mag, left), 10 μm (b, high magnification, right), and 5 μm (c).

    Journal: bioRxiv

    Article Title: APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

    doi: 10.1101/2020.09.18.303016

    Figure Lengend Snippet: Immunofluorescence analysis of paraffin-embedded sections of APC mutant mouse intestines. ( a ) HE-stained section of the intestines of an 16-weeks old Apc Δ716 mouse. Polyps analyzed in this study are indicated by numbers. ( b ) Immunostaining of a formalin-fixed, paraffin-embedded Apc Δ716 mouse intestine tissue section for E-cadherin and β-catenin with nuclear DNA stained by Hoechst 33342 to visualize crypt cells (top) and tumour cells (bottom) is shown. Low magnification images are shown on the left, and magnified images of the boxed areas are shown in the four panels on the right. Note that β-catenin levels are upregulated but its preferential accumulation to the nucleus is not observed. (c) Upregulation of TPX2 in Apc Δ716 tumour tissues. Immunostaining of a paraffin-embedded section for TPX2 with nuclear DNA stained by Hoechst 33342 is shown. Crypt cells (top) and tumour cells (bottom) were observed. Scale bars: 50 μm (b, low mag, left), 10 μm (b, high magnification, right), and 5 μm (c).

    Article Snippet: Antigen retrieval for β-catenin (610154, BD), E-cadherin (ECCD-2), AURKA, phosphor-AURKA(T288), TPX2, Ki67 and anti-α-tubulin (YL1/2) immunostaining was performed by boiling sections for 30 min in Tris-EDTA pH 9.0.

    Techniques: Immunofluorescence, Mutagenesis, Staining, Immunostaining, Formalin-fixed Paraffin-Embedded

    Characterization of cells used in this study. ( a ) Western blot analysis of HeLa cells and HeLa cells expressing EB1-GFP and H2B-TagRFP (clone A1) 31 before and after constitutive transfection with shRNA against APC (APC KD). APC knockdown efficiency was ∼70%. ( b ) Characterization of MCF10A cells, APC883 cells, and APC883 cell-derived transfectants. Cell lysates were subjected to western blotting using the indicated antibodies. Full length APC and truncated APC883 protein are indicated. For Myc-AURKA (T288D) and Myc-TPX2, endogenous and exogenous Myc-tagged proteins are indicated. ( c ) Characterization of MCF10A cells transfected with Myc-tagged wild-type β-catenin (Myc-WTβ-catenin) and a dominant stable β-catenin (Myc-MMβ-catenin), and further transfected with GFP-AURKA for fluorescence recovery after photobleaching (FRAP) analysis. Myc-WTβ-catenin- and Myc-MMβ-catenin-transfected cells were established under the same conditions, but Myc-WTβ-catenin was hardly overexpressed.

    Journal: bioRxiv

    Article Title: APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

    doi: 10.1101/2020.09.18.303016

    Figure Lengend Snippet: Characterization of cells used in this study. ( a ) Western blot analysis of HeLa cells and HeLa cells expressing EB1-GFP and H2B-TagRFP (clone A1) 31 before and after constitutive transfection with shRNA against APC (APC KD). APC knockdown efficiency was ∼70%. ( b ) Characterization of MCF10A cells, APC883 cells, and APC883 cell-derived transfectants. Cell lysates were subjected to western blotting using the indicated antibodies. Full length APC and truncated APC883 protein are indicated. For Myc-AURKA (T288D) and Myc-TPX2, endogenous and exogenous Myc-tagged proteins are indicated. ( c ) Characterization of MCF10A cells transfected with Myc-tagged wild-type β-catenin (Myc-WTβ-catenin) and a dominant stable β-catenin (Myc-MMβ-catenin), and further transfected with GFP-AURKA for fluorescence recovery after photobleaching (FRAP) analysis. Myc-WTβ-catenin- and Myc-MMβ-catenin-transfected cells were established under the same conditions, but Myc-WTβ-catenin was hardly overexpressed.

    Article Snippet: Antigen retrieval for β-catenin (610154, BD), E-cadherin (ECCD-2), AURKA, phosphor-AURKA(T288), TPX2, Ki67 and anti-α-tubulin (YL1/2) immunostaining was performed by boiling sections for 30 min in Tris-EDTA pH 9.0.

    Techniques: Western Blot, Expressing, Transfection, shRNA, Derivative Assay, Fluorescence

    Biochemical analysis of AURKA regulation by APC and AXIN1. ( a ) AXIN1 structure and AURKA-activating region that also binds to β-catenin 37 . ( b ) Effects of AXIN1 on AURKA phosphorylation were analyzed using the HEK293T cell overexpression assay system. HEK293T cells were transfected with GFP-fused AURKA and full length AXIN1 or fragments, lysed, and then subjected to western blotting using anti-GFP and anti-p-T288 AURKA antibodies. ( c ) Analysis of primary MEFs from wild-type (WT) and Apc 1638T mice. WB analysis of phospho-to-total AURKA ratio, and the immunostaining intensity of phospho-to-total AURKA ratio and γ-tubulin at centrosomes were normalized to those of the wild-type, which were set to 1.0 (c). Data are shown as means ± SE; ***P

    Journal: bioRxiv

    Article Title: APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

    doi: 10.1101/2020.09.18.303016

    Figure Lengend Snippet: Biochemical analysis of AURKA regulation by APC and AXIN1. ( a ) AXIN1 structure and AURKA-activating region that also binds to β-catenin 37 . ( b ) Effects of AXIN1 on AURKA phosphorylation were analyzed using the HEK293T cell overexpression assay system. HEK293T cells were transfected with GFP-fused AURKA and full length AXIN1 or fragments, lysed, and then subjected to western blotting using anti-GFP and anti-p-T288 AURKA antibodies. ( c ) Analysis of primary MEFs from wild-type (WT) and Apc 1638T mice. WB analysis of phospho-to-total AURKA ratio, and the immunostaining intensity of phospho-to-total AURKA ratio and γ-tubulin at centrosomes were normalized to those of the wild-type, which were set to 1.0 (c). Data are shown as means ± SE; ***P

    Article Snippet: Antigen retrieval for β-catenin (610154, BD), E-cadherin (ECCD-2), AURKA, phosphor-AURKA(T288), TPX2, Ki67 and anti-α-tubulin (YL1/2) immunostaining was performed by boiling sections for 30 min in Tris-EDTA pH 9.0.

    Techniques: Over Expression, Transfection, Western Blot, Mouse Assay, Immunostaining

    Immunofluorescence analysis of paraffin-embedded sections of APC mutant human tumor tissues. ( a ) HE-stained sections of a human hyperplasia polyp without APC mutation (polyp #1) (left) and a low grade tubular adenoma containing two APC truncation mutations (polyp #2) (right). Two mutant APC sequences detected in polyp #2 are shown (arrows). For details, see also SI Materials and Methods. ( b ) Ki67 proliferation index of the boxed area in (a) in the polyp #2. Immunofluorescence staining of polyp #2 for E-cadherin and Ki67. Nuclear DNA was stained with Hoechst 33342. Based on the Ki67 intensity, the area was roughly divided into a highly proliferative region (outer, high Ki67 area) and a moderately proliferative region (inner, low Ki67 area) as indicated by the yellow line. ( c ) Immunofluorescence staining of polyp #2 for TPX2 with Hoechst 33342 is shown. Examples of mitotic cells in less proliferative area (low Ki67) and highly proliferative area (high Ki67) shown in (b). ( d ) Immunostaining of the polyp #2 for E-cadherin and β-catenin with nuclear DNA stained by Hoechst 33342 is shown. Low magnification images are shown on the left, and magnified images of the boxed areas are shown in the four panels on the right. Note that preferential accumulation of β-catenin to the nucleus is not observed. Scale bars: 1 mm (a), 200 μm (b), 5 μm (c), 50 μm (d, left) and 20 μm (d, right).

    Journal: bioRxiv

    Article Title: APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

    doi: 10.1101/2020.09.18.303016

    Figure Lengend Snippet: Immunofluorescence analysis of paraffin-embedded sections of APC mutant human tumor tissues. ( a ) HE-stained sections of a human hyperplasia polyp without APC mutation (polyp #1) (left) and a low grade tubular adenoma containing two APC truncation mutations (polyp #2) (right). Two mutant APC sequences detected in polyp #2 are shown (arrows). For details, see also SI Materials and Methods. ( b ) Ki67 proliferation index of the boxed area in (a) in the polyp #2. Immunofluorescence staining of polyp #2 for E-cadherin and Ki67. Nuclear DNA was stained with Hoechst 33342. Based on the Ki67 intensity, the area was roughly divided into a highly proliferative region (outer, high Ki67 area) and a moderately proliferative region (inner, low Ki67 area) as indicated by the yellow line. ( c ) Immunofluorescence staining of polyp #2 for TPX2 with Hoechst 33342 is shown. Examples of mitotic cells in less proliferative area (low Ki67) and highly proliferative area (high Ki67) shown in (b). ( d ) Immunostaining of the polyp #2 for E-cadherin and β-catenin with nuclear DNA stained by Hoechst 33342 is shown. Low magnification images are shown on the left, and magnified images of the boxed areas are shown in the four panels on the right. Note that preferential accumulation of β-catenin to the nucleus is not observed. Scale bars: 1 mm (a), 200 μm (b), 5 μm (c), 50 μm (d, left) and 20 μm (d, right).

    Article Snippet: Antigen retrieval for β-catenin (610154, BD), E-cadherin (ECCD-2), AURKA, phosphor-AURKA(T288), TPX2, Ki67 and anti-α-tubulin (YL1/2) immunostaining was performed by boiling sections for 30 min in Tris-EDTA pH 9.0.

    Techniques: Immunofluorescence, Mutagenesis, Staining, Immunostaining

    APC structure and phenotypes. ( a ) Domain structures of full length APC and major binding partners discussed in this study 29 , 40 , 41 , 86– 89 . The APC gene is responsible for many sporadic cases of gastrointestinal cancers and familial adenomatous polyposis (FAP), a dominantly inherited colorectal tumour predisposition that results from germline mutations in APC 21 , 22 , 90– 94 . Tumours start developing upon mutation in both alleles. In general, APC mutations that result in stable proteins truncated in the region between the armadillo repeat and first SAMP motif are associated with severe polyposis phenotypes (reviewed in 40, 95). ( b ) Truncated APC mutants. Phenotypes of cells and mice are shown on the right. Heterozygous Apc mutations, such as Apc Min and Apc Δ7 16 , leads to severe polyposis after loss of the wild-type allele ( > ∼30 polyps), whereas their homozygosity results in embryonic lethality 46 , 96 , 97 . Homozygous Apc 1638T mice harboring a truncating mutation just after the first SAMP motif, which retains the β-catenin destruction ability, are viable and do not develop any tumours, demonstrating that this N-terminal fragment retaining the first SAMP motif is sufficient for the tumour-suppressing function 39 . Nevertheless, Apc 1638T homozygous ES cells present a CIN phenotype, although it is weaker than that in Apc Min homozygous ES cells 16 . MCF10A-derived cell line APC883 harbouring a homozygous APC truncation was generated by genome editing.

    Journal: bioRxiv

    Article Title: APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

    doi: 10.1101/2020.09.18.303016

    Figure Lengend Snippet: APC structure and phenotypes. ( a ) Domain structures of full length APC and major binding partners discussed in this study 29 , 40 , 41 , 86– 89 . The APC gene is responsible for many sporadic cases of gastrointestinal cancers and familial adenomatous polyposis (FAP), a dominantly inherited colorectal tumour predisposition that results from germline mutations in APC 21 , 22 , 90– 94 . Tumours start developing upon mutation in both alleles. In general, APC mutations that result in stable proteins truncated in the region between the armadillo repeat and first SAMP motif are associated with severe polyposis phenotypes (reviewed in 40, 95). ( b ) Truncated APC mutants. Phenotypes of cells and mice are shown on the right. Heterozygous Apc mutations, such as Apc Min and Apc Δ7 16 , leads to severe polyposis after loss of the wild-type allele ( > ∼30 polyps), whereas their homozygosity results in embryonic lethality 46 , 96 , 97 . Homozygous Apc 1638T mice harboring a truncating mutation just after the first SAMP motif, which retains the β-catenin destruction ability, are viable and do not develop any tumours, demonstrating that this N-terminal fragment retaining the first SAMP motif is sufficient for the tumour-suppressing function 39 . Nevertheless, Apc 1638T homozygous ES cells present a CIN phenotype, although it is weaker than that in Apc Min homozygous ES cells 16 . MCF10A-derived cell line APC883 harbouring a homozygous APC truncation was generated by genome editing.

    Article Snippet: Antigen retrieval for β-catenin (610154, BD), E-cadherin (ECCD-2), AURKA, phosphor-AURKA(T288), TPX2, Ki67 and anti-α-tubulin (YL1/2) immunostaining was performed by boiling sections for 30 min in Tris-EDTA pH 9.0.

    Techniques: Binding Assay, Mutagenesis, Mouse Assay, Derivative Assay, Generated

    Karyotypes of MCF10A and APC883 cells. Representative karyotype images of each cell line (20 cells were analyzed in each cell line). For complete karyotype descriptions of all analyzed cells, see Table 1 . The karyotypes were determined by Q-banding with Hoechst/quinacrine staining. Note that commonly observed abnormalities in chromosome 1 (deletion and partial duplication), chromosome 3 (deletion and translocation), chromosome 8 (duplication), and chromosome 9 (translocation) have been observed previously 81 , 82 . ( a ) Karyotype of parental MCF10A cells expressing full length APC. The MCF10A cell line used has two major populations with or without chromosome 20 trisomy (types 1 and 2, respectively), whereas all 20 cells showed near-diploidy. Chromosome 20 is indicated by red arrows. ( b – f ) Karyotypes of APC883 cells and APC883 cell-derived cells. Major karyotypes in each cell type (c, d) and representative examples carrying chromosome aberrations (b, e, and f) are shown. Newly derived abnormal chromosomes are indicated by red arrowheads. After reduction of β-catenin levels (c) or expression of constitutively active AURKA (T288D) (d), APC883 cells maintained their original karyotype. In TPX2-expressing APC883 cells (e), various aberrations were observed. In (f), representative karyotypes of APC883 cells after > 5 months passages are shown. In this subpopulation, all cells analyzed had near-tetraploid karyotype with some numerical imbalances. Common numerical imbalances are shown by the letter color for the karyotype description: cyan, three chromosome 6s; brown, five chromosome 8s; red, six chromosome 20s. Examples of randomly occurring imbalances are indicated by arrows.

    Journal: bioRxiv

    Article Title: APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

    doi: 10.1101/2020.09.18.303016

    Figure Lengend Snippet: Karyotypes of MCF10A and APC883 cells. Representative karyotype images of each cell line (20 cells were analyzed in each cell line). For complete karyotype descriptions of all analyzed cells, see Table 1 . The karyotypes were determined by Q-banding with Hoechst/quinacrine staining. Note that commonly observed abnormalities in chromosome 1 (deletion and partial duplication), chromosome 3 (deletion and translocation), chromosome 8 (duplication), and chromosome 9 (translocation) have been observed previously 81 , 82 . ( a ) Karyotype of parental MCF10A cells expressing full length APC. The MCF10A cell line used has two major populations with or without chromosome 20 trisomy (types 1 and 2, respectively), whereas all 20 cells showed near-diploidy. Chromosome 20 is indicated by red arrows. ( b – f ) Karyotypes of APC883 cells and APC883 cell-derived cells. Major karyotypes in each cell type (c, d) and representative examples carrying chromosome aberrations (b, e, and f) are shown. Newly derived abnormal chromosomes are indicated by red arrowheads. After reduction of β-catenin levels (c) or expression of constitutively active AURKA (T288D) (d), APC883 cells maintained their original karyotype. In TPX2-expressing APC883 cells (e), various aberrations were observed. In (f), representative karyotypes of APC883 cells after > 5 months passages are shown. In this subpopulation, all cells analyzed had near-tetraploid karyotype with some numerical imbalances. Common numerical imbalances are shown by the letter color for the karyotype description: cyan, three chromosome 6s; brown, five chromosome 8s; red, six chromosome 20s. Examples of randomly occurring imbalances are indicated by arrows.

    Article Snippet: Antigen retrieval for β-catenin (610154, BD), E-cadherin (ECCD-2), AURKA, phosphor-AURKA(T288), TPX2, Ki67 and anti-α-tubulin (YL1/2) immunostaining was performed by boiling sections for 30 min in Tris-EDTA pH 9.0.

    Techniques: Staining, Translocation Assay, Expressing, Derivative Assay

    Association of four epithelial-mesenchymal transition-a ssociated markers with overall survival. Kaplan-Meier overall survival curves stratified by the expression of (A) E-cadherin, (B) N-cadherin, (C) β-catenin and (D) ZEB2. P-values were calculated using the log-rank test. ZEB2, zinc finger E-box binding homeobox 2.

    Journal: Oncology Letters

    Article Title: Role of epithelial-mesenchymal transition markers E-cadherin, N-cadherin, β-catenin and ZEB2 in laryngeal squamous cell carcinoma

    doi: 10.3892/ol.2018.7751

    Figure Lengend Snippet: Association of four epithelial-mesenchymal transition-a ssociated markers with overall survival. Kaplan-Meier overall survival curves stratified by the expression of (A) E-cadherin, (B) N-cadherin, (C) β-catenin and (D) ZEB2. P-values were calculated using the log-rank test. ZEB2, zinc finger E-box binding homeobox 2.

    Article Snippet: Sections were subsequently incubated with primary antibodies against E-cadherin (mouse monoclonal; cat. no., ab1416), N-cadherin (rabbit polyclonal; cat. no., ab18203), β-catenin (rabbit monoclonal; cat. no., ab32572), and ZEB2 (rabbit polyclonal; cat. no., ab138222; all Abcam, Cambridge, UK) overnight at 4°C.

    Techniques: Expressing, Binding Assay

    Analysis of mRNA expression of Wnt and β-catenin by reverse transcription-quantitative polymerase chain reaction, following miR-184 inhibition and overexpression. (A) Wnt and β-catenin mRNA expression in U-2OS cells. (B) Wnt and β-catenin mRNA expression in 143B cells. *P

    Journal: Molecular Medicine Reports

    Article Title: Regulatory effects of microRNA-184 on osteosarcoma via the Wnt/β-catenin signaling pathway

    doi: 10.3892/mmr.2018.9184

    Figure Lengend Snippet: Analysis of mRNA expression of Wnt and β-catenin by reverse transcription-quantitative polymerase chain reaction, following miR-184 inhibition and overexpression. (A) Wnt and β-catenin mRNA expression in U-2OS cells. (B) Wnt and β-catenin mRNA expression in 143B cells. *P

    Article Snippet: Primary antibodies were as follows: Anti-Wnt (cat. no. ab15251; 1:1,000), anti-β-catenin (cat. no. ab32572; 1:3,000), anti-phosphorylated-β-catenin (cat. no. ab27798; 1:500) and anti-GAPDH (cat. no. ab181602; 1:1,000; all Abcam, Cambridge, UK).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Inhibition, Over Expression

    Protein expressions of Wnt pathway molecules after downregulation of Gal-3 in ovarian cancer cell lines. Notes: Downregulating Gal-3 expression attenuates β-catenin expression in ovarian epithelial cancer cells SKOV3 and OVCAR3; c-myc and cyclin D1 which are both downstream proteins in Wnt pathways are downregulated. Abbreviations: Gal-3, galectin-3; NC, negative control; siRNA, small interfering RNA.

    Journal: Cancer Management and Research

    Article Title: Galectin-3 and β-catenin are associated with a poor prognosis in serous epithelial ovarian cancer

    doi: 10.2147/CMAR.S171146

    Figure Lengend Snippet: Protein expressions of Wnt pathway molecules after downregulation of Gal-3 in ovarian cancer cell lines. Notes: Downregulating Gal-3 expression attenuates β-catenin expression in ovarian epithelial cancer cells SKOV3 and OVCAR3; c-myc and cyclin D1 which are both downstream proteins in Wnt pathways are downregulated. Abbreviations: Gal-3, galectin-3; NC, negative control; siRNA, small interfering RNA.

    Article Snippet: Sections were then incubated with anti-Gal-3 (mouse anti-Gal-3 monoclonal antibody; 1:150 dilution; Abcam, Cambridge, UK) and anti-β-catenin (rabbit β-catenin monoclonal antibody; 1:100 dilution; Cell Signaling Technology, Danvers, MA, USA) antibodies overnight at 4°C.

    Techniques: Expressing, Negative Control, Small Interfering RNA

    CK1ε depletion induces apoptosis and growth inhibition through activating β-catenin. ( A ) β-catenin signaling in CK1ε-deficient U87MG cells. Phosphorylated, active, and total β-catenin was assessed using immunoblotting. ACTB (β-actin) is the loading control. Band intensities were quantified using Image J. ( B ) β-catenin signaling in CK1ε-deficient A172 cells. ( C ) Luciferase reporter assay. The TOPFlash plasmid harbors TCF/LEF binding sites and responds to β-catenin activation. FOPFlash contains mutated TCF/LEF binding sites and does not respond to β-catenin activation. ( D ) Immunoblotting of cleaved caspase 3 (c-CASP3, an apoptosis marker) and LC3B (an autophagy marker). ( E ) Caspase 3/7 activity assay. ( F ) Knockdown of β-catenin. U87MG cells were transduced with viruses of NS shRNA or β-catenin shRNA. ( G ) Viability of U87MG cells treated with CK1ε shRNA and/or β-catenin shRNA. ( H ) Immunoblotting of c-CASP3 in U87MG cells upon depletion of CK1ε and/or β-catenin. Error bars represent standard deviations from three independent experiments. Full length blots were presented in supplemental materials. * P

    Journal: Scientific Reports

    Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

    doi: 10.1038/s41598-018-31864-x

    Figure Lengend Snippet: CK1ε depletion induces apoptosis and growth inhibition through activating β-catenin. ( A ) β-catenin signaling in CK1ε-deficient U87MG cells. Phosphorylated, active, and total β-catenin was assessed using immunoblotting. ACTB (β-actin) is the loading control. Band intensities were quantified using Image J. ( B ) β-catenin signaling in CK1ε-deficient A172 cells. ( C ) Luciferase reporter assay. The TOPFlash plasmid harbors TCF/LEF binding sites and responds to β-catenin activation. FOPFlash contains mutated TCF/LEF binding sites and does not respond to β-catenin activation. ( D ) Immunoblotting of cleaved caspase 3 (c-CASP3, an apoptosis marker) and LC3B (an autophagy marker). ( E ) Caspase 3/7 activity assay. ( F ) Knockdown of β-catenin. U87MG cells were transduced with viruses of NS shRNA or β-catenin shRNA. ( G ) Viability of U87MG cells treated with CK1ε shRNA and/or β-catenin shRNA. ( H ) Immunoblotting of c-CASP3 in U87MG cells upon depletion of CK1ε and/or β-catenin. Error bars represent standard deviations from three independent experiments. Full length blots were presented in supplemental materials. * P

    Article Snippet: Antibody of active β-catenin was purchased from EMD Millipore Corporation.

    Techniques: Inhibition, Luciferase, Reporter Assay, Plasmid Preparation, Binding Assay, Activation Assay, Marker, Activity Assay, Transduction, shRNA

    A noncanonical CK1ε/β-catenin signaling in GBM cell survival.

    Journal: Scientific Reports

    Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

    doi: 10.1038/s41598-018-31864-x

    Figure Lengend Snippet: A noncanonical CK1ε/β-catenin signaling in GBM cell survival.

    Article Snippet: Antibody of active β-catenin was purchased from EMD Millipore Corporation.

    Techniques:

    CK1ε regulates β-catenin activity and self-renewal of GSCs. ( A ). ( B ) Viability of LN-18/GSC, LN229/GSCs, and U251/GSC upon depletion of CK1ε or MELK. ( C ) Images of GS9-6/NOTCH1 spheres treated with NS, CK1ε, or MELK shRNA. Scale bar: 25 μm. ( D ) Self-renewal assay of VTC-001/GSC, GS9-6/NOTCH1, and LN-18/GSC treated with NS, CK1ε, or MELK shRNA. Percentages of wells with spheres represent capabilities of GSCs to self-renew. ( E ) Differentiation of GS9-6/NOTCH1 cells. Cells were treated with fetal bovine serum (FBS) to induce differentiation, which was indicated by elevated levels of GFAP (an astrocyte marker). ( F ) Immunoblotting of active β-catenin, β-catenin, c-CASP3, NOTCH1, and GFAP in CK1ε-deficient GS9-6/NOTCH1 cells. ( G ) Immunoblotting of active β-catenin, β-catenin, or c-CASP3 in CK1ε-deficient LN229/GSCs. Full length blots were presented in supplemental materials. * P

    Journal: Scientific Reports

    Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

    doi: 10.1038/s41598-018-31864-x

    Figure Lengend Snippet: CK1ε regulates β-catenin activity and self-renewal of GSCs. ( A ). ( B ) Viability of LN-18/GSC, LN229/GSCs, and U251/GSC upon depletion of CK1ε or MELK. ( C ) Images of GS9-6/NOTCH1 spheres treated with NS, CK1ε, or MELK shRNA. Scale bar: 25 μm. ( D ) Self-renewal assay of VTC-001/GSC, GS9-6/NOTCH1, and LN-18/GSC treated with NS, CK1ε, or MELK shRNA. Percentages of wells with spheres represent capabilities of GSCs to self-renew. ( E ) Differentiation of GS9-6/NOTCH1 cells. Cells were treated with fetal bovine serum (FBS) to induce differentiation, which was indicated by elevated levels of GFAP (an astrocyte marker). ( F ) Immunoblotting of active β-catenin, β-catenin, c-CASP3, NOTCH1, and GFAP in CK1ε-deficient GS9-6/NOTCH1 cells. ( G ) Immunoblotting of active β-catenin, β-catenin, or c-CASP3 in CK1ε-deficient LN229/GSCs. Full length blots were presented in supplemental materials. * P

    Article Snippet: Antibody of active β-catenin was purchased from EMD Millipore Corporation.

    Techniques: Activity Assay, shRNA, Marker

    The CK1ε inhibitor IC261 blocks GBM cell growth with no effect on astrocytes. ( A ) IC50 of IC261 in U87MG cells. U87MG cells were treated with IC261 at various doses. Viability was determined using the MTS assay and IC50 was calculated using Prism. ( B ) IC50 of PF-4800567 in U87MG cells. ( C ) Cytotoxicity of IC261 in astrocytes and nine GBM cell lines. ( D ) Immunoblotting of CK1ε/β-catenin signaling in U87MG cells treated with IC261 or PF-4800567. Full length blots were presented in supplemental materials. * P

    Journal: Scientific Reports

    Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

    doi: 10.1038/s41598-018-31864-x

    Figure Lengend Snippet: The CK1ε inhibitor IC261 blocks GBM cell growth with no effect on astrocytes. ( A ) IC50 of IC261 in U87MG cells. U87MG cells were treated with IC261 at various doses. Viability was determined using the MTS assay and IC50 was calculated using Prism. ( B ) IC50 of PF-4800567 in U87MG cells. ( C ) Cytotoxicity of IC261 in astrocytes and nine GBM cell lines. ( D ) Immunoblotting of CK1ε/β-catenin signaling in U87MG cells treated with IC261 or PF-4800567. Full length blots were presented in supplemental materials. * P

    Article Snippet: Antibody of active β-catenin was purchased from EMD Millipore Corporation.

    Techniques: MTS Assay

    Aspirin treatment increases expression of intracellular DKK-1 while reducing secreted DKK-1. ( A ) Immunoblotting for DKK-1 and LRP6 protein abundance in whole cell extracts and secreted DKK-1 protein abundance in media from HCT116 cells treated with 0.5 mmol/L aspirin, 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 and 24 hours. ( B ) Immunoblotting for DKK-1 and LRP6 protein abundance in whole cell extracts and secreted DKK-1 protein abundance in media from Colo205 cells treated with 0.5 mmol/L aspirin, 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 and 24 hours. ( C ) Immunoblotting of DKK-1, c-myc, and Snail protein abundance in cytoplasmic/nuclear extracts from HCT116 cells treated with 3 mmol/L aspirin for 24 hours. Immunoblotting data representative of 3 independent experiments. ( D ) DKK-1 transcript expression in organoids derived from human FAP normal colonic mucosa tissue and adenomatous tissue treated with 2 mmol/L aspirin for 4 hours. DKK-1 transcript levels are normalized to 18S transcripts, and data are expressed as fold-change compared with untreated control sample. DKK-1 transcript data represent 3 individual FAP patient samples. ( E ) DKK-1 transcript expression in fetal organoids treated with 2 mmol/L aspirin for 8 days. DKK-1 transcript levels are normalized to 18S transcripts, and data are expressed as fold-change compared with untreated control sample. ( F ) Average size of fetal organoids treated with 2 mmol/L aspirin for 8 days. Fetal organoid data represent 4 independent experiments. (G) Immunoblotting of DKK-1, β-catenin, E-cadherin, SOX-9, c-myc, and Snail protein abundance in untransfected HCT116 cells and HCT116 cells transfected with siRNA targeting DKK-1 or control siRNA. Cells were treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours. β-actin represents sample control. DKK-1 siRNA data representative of 3 independent experiments. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by Student t test. Asterisks denote P value (∗

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1

    doi: 10.1016/j.jcmgh.2020.09.010

    Figure Lengend Snippet: Aspirin treatment increases expression of intracellular DKK-1 while reducing secreted DKK-1. ( A ) Immunoblotting for DKK-1 and LRP6 protein abundance in whole cell extracts and secreted DKK-1 protein abundance in media from HCT116 cells treated with 0.5 mmol/L aspirin, 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 and 24 hours. ( B ) Immunoblotting for DKK-1 and LRP6 protein abundance in whole cell extracts and secreted DKK-1 protein abundance in media from Colo205 cells treated with 0.5 mmol/L aspirin, 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 and 24 hours. ( C ) Immunoblotting of DKK-1, c-myc, and Snail protein abundance in cytoplasmic/nuclear extracts from HCT116 cells treated with 3 mmol/L aspirin for 24 hours. Immunoblotting data representative of 3 independent experiments. ( D ) DKK-1 transcript expression in organoids derived from human FAP normal colonic mucosa tissue and adenomatous tissue treated with 2 mmol/L aspirin for 4 hours. DKK-1 transcript levels are normalized to 18S transcripts, and data are expressed as fold-change compared with untreated control sample. DKK-1 transcript data represent 3 individual FAP patient samples. ( E ) DKK-1 transcript expression in fetal organoids treated with 2 mmol/L aspirin for 8 days. DKK-1 transcript levels are normalized to 18S transcripts, and data are expressed as fold-change compared with untreated control sample. ( F ) Average size of fetal organoids treated with 2 mmol/L aspirin for 8 days. Fetal organoid data represent 4 independent experiments. (G) Immunoblotting of DKK-1, β-catenin, E-cadherin, SOX-9, c-myc, and Snail protein abundance in untransfected HCT116 cells and HCT116 cells transfected with siRNA targeting DKK-1 or control siRNA. Cells were treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours. β-actin represents sample control. DKK-1 siRNA data representative of 3 independent experiments. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by Student t test. Asterisks denote P value (∗

    Article Snippet: For each reaction 5 μL of anti-E-cadherin (BD Transduction Laboratories, 610181) or anti-β-catenin (BD Transduction Laboratories, 610153) antibody was added to 800 μg protein and incubated at 4°C for 24 hours.

    Techniques: Expressing, Derivative Assay, Transfection, Standard Deviation

    Aspirin reduces β-catenin and EMT markers in vivo. ( A ) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) Apc Min/+ mouse tissue stained for β-catenin expression. ( B ) Transcript expression of Tcf7 in Apc Min/+ mouse crypts treated with 400 mg/kg aspirin for 4 weeks and Apc flox/flox mouse organoids treated with 2 mmol/L aspirin for 12 days. Tcf7 transcript levels are normalized to GAPDH transcripts and expressed as fold-change compared with untreated control sample. Data represent 1 mouse per experimental condition. ( C ) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) Apc Min/+ mouse tissue stained for E-cadherin expression. ( D ) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) Apc Min/+ mouse tissue stained for vimentin expression. Quantification of β-catenin (21 control and 30 aspirin-treated adenomas), E-cadherin (37 control and 23 aspirin-treated adenomas), and vimentin (29 control and 16 aspirin-treated adenomas) staining in adenoma tissue from cohort of 5 Apc Min/+ control and 4 Apc Min/+ aspirin-treated mice. Microscope objective magnification noted in bottom left corner of image. Scale bar = 50 μm. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by unpaired Student t test. Asterisks denote P value (∗

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1

    doi: 10.1016/j.jcmgh.2020.09.010

    Figure Lengend Snippet: Aspirin reduces β-catenin and EMT markers in vivo. ( A ) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) Apc Min/+ mouse tissue stained for β-catenin expression. ( B ) Transcript expression of Tcf7 in Apc Min/+ mouse crypts treated with 400 mg/kg aspirin for 4 weeks and Apc flox/flox mouse organoids treated with 2 mmol/L aspirin for 12 days. Tcf7 transcript levels are normalized to GAPDH transcripts and expressed as fold-change compared with untreated control sample. Data represent 1 mouse per experimental condition. ( C ) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) Apc Min/+ mouse tissue stained for E-cadherin expression. ( D ) Immunohistochemistry images and quantification of control and aspirin-treated (400 mg/kg aspirin by oral gavage for 4 weeks) Apc Min/+ mouse tissue stained for vimentin expression. Quantification of β-catenin (21 control and 30 aspirin-treated adenomas), E-cadherin (37 control and 23 aspirin-treated adenomas), and vimentin (29 control and 16 aspirin-treated adenomas) staining in adenoma tissue from cohort of 5 Apc Min/+ control and 4 Apc Min/+ aspirin-treated mice. Microscope objective magnification noted in bottom left corner of image. Scale bar = 50 μm. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by unpaired Student t test. Asterisks denote P value (∗

    Article Snippet: For each reaction 5 μL of anti-E-cadherin (BD Transduction Laboratories, 610181) or anti-β-catenin (BD Transduction Laboratories, 610153) antibody was added to 800 μg protein and incubated at 4°C for 24 hours.

    Techniques: In Vivo, Immunohistochemistry, Staining, Expressing, Mouse Assay, Microscopy, Standard Deviation

    Aspirin reduces Wnt-driven mesenchymal and stem marker expression in CRC cell lines and FAP organoids. ( A ) Immunoblotting of phospho-β-catenin (S33/S37/T41), β-catenin, phospho-GSK3β (S9), and GSK3β protein abundance in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 hours. ( B ) Immunoblotting of β-catenin, Axin2, and c-myc protein abundance in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours. ( C ) Immunoblotting of E-cadherin, Snail, and Lgr5 protein abundance in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours. Immunoblotting data representative of 3 independent experiments. ( D1 ) Immunoprecipitation using E-cadherin antibody in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours and immunoblotting of β-catenin and E-cadherin from input lysates and immunoprecipitation elutes. ( D2 ) Immunoprecipitation using β-catenin antibody in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours and immunoblotting of β-catenin and E-cadherin from input lysates and immunoprecipitation elutes. Immunoprecipitation data representative of 3 independent experiments. ( E ) Quantification of percentage of budding organoids derived from human FAP patient normal colonic mucosa tissue treated with 0.5 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 15 days in vitro. Organoid data represent 1 individual human patient sample. ( F ) Immunoblotting of β-catenin, SOX-9, and Lgr5 protein abundance in organoids derived from human FAP patient normal colonic mucosa tissue treated with 0.5 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 36 days in vitro. Organoid data represent 1 individual human patient sample. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by Student t test. Asterisks denote P value (∗

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1

    doi: 10.1016/j.jcmgh.2020.09.010

    Figure Lengend Snippet: Aspirin reduces Wnt-driven mesenchymal and stem marker expression in CRC cell lines and FAP organoids. ( A ) Immunoblotting of phospho-β-catenin (S33/S37/T41), β-catenin, phospho-GSK3β (S9), and GSK3β protein abundance in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 16 hours. ( B ) Immunoblotting of β-catenin, Axin2, and c-myc protein abundance in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours. ( C ) Immunoblotting of E-cadherin, Snail, and Lgr5 protein abundance in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours. Immunoblotting data representative of 3 independent experiments. ( D1 ) Immunoprecipitation using E-cadherin antibody in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours and immunoblotting of β-catenin and E-cadherin from input lysates and immunoprecipitation elutes. ( D2 ) Immunoprecipitation using β-catenin antibody in HCT116 cells treated with 3 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 24 hours and immunoblotting of β-catenin and E-cadherin from input lysates and immunoprecipitation elutes. Immunoprecipitation data representative of 3 independent experiments. ( E ) Quantification of percentage of budding organoids derived from human FAP patient normal colonic mucosa tissue treated with 0.5 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 15 days in vitro. Organoid data represent 1 individual human patient sample. ( F ) Immunoblotting of β-catenin, SOX-9, and Lgr5 protein abundance in organoids derived from human FAP patient normal colonic mucosa tissue treated with 0.5 mmol/L aspirin, 3 μmol/L CHIR-99021, or combination for 36 days in vitro. Organoid data represent 1 individual human patient sample. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by Student t test. Asterisks denote P value (∗

    Article Snippet: For each reaction 5 μL of anti-E-cadherin (BD Transduction Laboratories, 610181) or anti-β-catenin (BD Transduction Laboratories, 610153) antibody was added to 800 μg protein and incubated at 4°C for 24 hours.

    Techniques: Marker, Expressing, Immunoprecipitation, Derivative Assay, In Vitro, Standard Deviation

    Aspirin reduces Wnt signaling and promotes an epithelial phenotype in CRC cells. ( A ) Immunoblotting of β-catenin, c-myc, Lgr5, and E-cadherin protein abundance in Colo205 cells treated with 0.5 or 3 mmol/L aspirin for 24 hours. ( B ) Immunoblotting of E-cadherin protein expression in HCT116 cells treated with 0.5 or 3 mmol/L aspirin for 24 hours. ( C ) E-cadherin (CDH1) transcript expression in HCT116 and Colo205 cells treated with 3 mmol/L aspirin for either 24 or 48 hours. CDH1 transcript levels are normalized to GAPDH transcripts and expressed as fold-change compared with untreated control sample. Data represent 3 independent experiments. ( D1 ) Immunofluorescence images of E-cadherin and zona occludens 1 staining in HCT116 cells treated with 0.5 mmol/L aspirin for 24 hours. ( D2 ) Quantification of E-cadherin and zona occludens 1 staining in HCT116 cells treated with 0.5 mmol/L aspirin for 24 hours. Staining intensity quantified as mean grey intensity per cell. Data represent 2 independent experiments. ( E ) Immunoblotting of E-cadherin and Snail protein abundance in cytoplasmic/nuclear extracts from HCT116 cells treated with 0.5 or 3 mmol/L aspirin for 16 hours. Immunoblotting data representative of 3 independent experiments. Microscope objective magnification noted in bottom left corner of image. Scale bar = 50 μm. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by unpaired Student t test. Asterisks denote P value (∗

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1

    doi: 10.1016/j.jcmgh.2020.09.010

    Figure Lengend Snippet: Aspirin reduces Wnt signaling and promotes an epithelial phenotype in CRC cells. ( A ) Immunoblotting of β-catenin, c-myc, Lgr5, and E-cadherin protein abundance in Colo205 cells treated with 0.5 or 3 mmol/L aspirin for 24 hours. ( B ) Immunoblotting of E-cadherin protein expression in HCT116 cells treated with 0.5 or 3 mmol/L aspirin for 24 hours. ( C ) E-cadherin (CDH1) transcript expression in HCT116 and Colo205 cells treated with 3 mmol/L aspirin for either 24 or 48 hours. CDH1 transcript levels are normalized to GAPDH transcripts and expressed as fold-change compared with untreated control sample. Data represent 3 independent experiments. ( D1 ) Immunofluorescence images of E-cadherin and zona occludens 1 staining in HCT116 cells treated with 0.5 mmol/L aspirin for 24 hours. ( D2 ) Quantification of E-cadherin and zona occludens 1 staining in HCT116 cells treated with 0.5 mmol/L aspirin for 24 hours. Staining intensity quantified as mean grey intensity per cell. Data represent 2 independent experiments. ( E ) Immunoblotting of E-cadherin and Snail protein abundance in cytoplasmic/nuclear extracts from HCT116 cells treated with 0.5 or 3 mmol/L aspirin for 16 hours. Immunoblotting data representative of 3 independent experiments. Microscope objective magnification noted in bottom left corner of image. Scale bar = 50 μm. Graphs represent individual data plots with overlay of mean and standard deviation. Statistical significance determined by unpaired Student t test. Asterisks denote P value (∗

    Article Snippet: For each reaction 5 μL of anti-E-cadherin (BD Transduction Laboratories, 610181) or anti-β-catenin (BD Transduction Laboratories, 610153) antibody was added to 800 μg protein and incubated at 4°C for 24 hours.

    Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Standard Deviation

    CHIR activates Wnt/β-catenin pathway in bladder cancer cell line-derived organoids ( A ) Bar graphs show the relative gene expression of AXIN2 by qRT-PCR in RT4- and 5637-derived organoids treated for 72 hours with DMSO or CHIR of 5 and 2.5 µM for RT4 and 5637, respectively. GAPDH was used as a reference gene, and relative expression was calculated by the 2 ΔΔCT method. ** P ≤ 0.01. ( B ) Western blot analysis shows protein levels of β-catenin, FOXA1, and GAPDH in RT4- and 5637-derived organoids. Organoids were treated for 24 hours with DMSO or CHIR of 5 and 2.5 µM for RT4 and 5637, respectively. Cytoplasmic and nuclear proteins were separated and analyzed. GAPDH and FOXA1 were used as a loading control for cytoplasmic and nuclear protein, respectively.

    Journal: Oncotarget

    Article Title: Three-dimensional organoid culture reveals involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells

    doi: 10.18632/oncotarget.24308

    Figure Lengend Snippet: CHIR activates Wnt/β-catenin pathway in bladder cancer cell line-derived organoids ( A ) Bar graphs show the relative gene expression of AXIN2 by qRT-PCR in RT4- and 5637-derived organoids treated for 72 hours with DMSO or CHIR of 5 and 2.5 µM for RT4 and 5637, respectively. GAPDH was used as a reference gene, and relative expression was calculated by the 2 ΔΔCT method. ** P ≤ 0.01. ( B ) Western blot analysis shows protein levels of β-catenin, FOXA1, and GAPDH in RT4- and 5637-derived organoids. Organoids were treated for 24 hours with DMSO or CHIR of 5 and 2.5 µM for RT4 and 5637, respectively. Cytoplasmic and nuclear proteins were separated and analyzed. GAPDH and FOXA1 were used as a loading control for cytoplasmic and nuclear protein, respectively.

    Article Snippet: Organoids were incubated at 4° C with anti-β-catenin antibody (610154, 1:500, Becton Dickinson) overnight, then with Alexa-488 conjugated secondary antibody (Thermo Fischer Scientific) for 1 hour.

    Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Western Blot

    CHIR enhances proliferation of primary bladder cancer cells in organoid culture with activation of Wnt/β-catenin pathway ( A ) Representative images of primary cell organoids (BCa #01) at day 1 and day 6 treated with DMSO or CHIR of 10 µM. Scale bar, 100 µm. ( B ) Relative growth of organoids from 4 patient samples was calculated by dividing area at day 6 by that at day 1, and depicted in the plot. ( C ) Relative viability of organoids from 2 patient samples was measured by dividing the amount of ATP at day 6 by the area of organoid at day 1, and depicted in the plot. Each dot represents relative growth or viability of one organoid. ( D ) Bar graphs show the relative gene expression of AXIN2 by qRT-PCR in primary cell organoids (BCa #04) treated with DMSO or CHIR of 10 µM for 96 hours. GAPDH was used as a reference gene, and relative expression was calculated by the 2 ΔΔCT method. ( E ) A bar graph shows the relative gene expression of AXIN2 by qRT-PCR in primary cell organoids of 3 pooled patient samples (BCa #02, #03, #04). The result was normalized to the expression of DMSO-treated organoids. * P ≤ 0.05. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001. ( F ) The expression and subcellular localization of β-catenin in primary cell organoids (BCa #04) treated with DMSO or 10 µM CHIR for 72 hours was detected by immunofluorescence staining. Nuclei and actin fibers were counterstained with DAPI and ActinRed, respectively. Scale bar 20 µm.

    Journal: Oncotarget

    Article Title: Three-dimensional organoid culture reveals involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells

    doi: 10.18632/oncotarget.24308

    Figure Lengend Snippet: CHIR enhances proliferation of primary bladder cancer cells in organoid culture with activation of Wnt/β-catenin pathway ( A ) Representative images of primary cell organoids (BCa #01) at day 1 and day 6 treated with DMSO or CHIR of 10 µM. Scale bar, 100 µm. ( B ) Relative growth of organoids from 4 patient samples was calculated by dividing area at day 6 by that at day 1, and depicted in the plot. ( C ) Relative viability of organoids from 2 patient samples was measured by dividing the amount of ATP at day 6 by the area of organoid at day 1, and depicted in the plot. Each dot represents relative growth or viability of one organoid. ( D ) Bar graphs show the relative gene expression of AXIN2 by qRT-PCR in primary cell organoids (BCa #04) treated with DMSO or CHIR of 10 µM for 96 hours. GAPDH was used as a reference gene, and relative expression was calculated by the 2 ΔΔCT method. ( E ) A bar graph shows the relative gene expression of AXIN2 by qRT-PCR in primary cell organoids of 3 pooled patient samples (BCa #02, #03, #04). The result was normalized to the expression of DMSO-treated organoids. * P ≤ 0.05. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001. ( F ) The expression and subcellular localization of β-catenin in primary cell organoids (BCa #04) treated with DMSO or 10 µM CHIR for 72 hours was detected by immunofluorescence staining. Nuclei and actin fibers were counterstained with DAPI and ActinRed, respectively. Scale bar 20 µm.

    Article Snippet: Organoids were incubated at 4° C with anti-β-catenin antibody (610154, 1:500, Becton Dickinson) overnight, then with Alexa-488 conjugated secondary antibody (Thermo Fischer Scientific) for 1 hour.

    Techniques: Activation Assay, BIA-KA, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

    β-catenin is required for growth of bladder cancer organoids ( A , D ) Western blot of β-catenin in RT4 and 5637 transiently transfected with siRNAs against β-catenin. Protein was extracted 48 hours after transfection. GAPDH was used as a loading control. NC, negative control. ( B ) RT4 was transiently transfected with siRNAs and used to generate organoids. Images show representative RT4-derived organoids after transfection at day 1 and 6. Scale bar, 100 µm. ( C ) Relative growth of RT4-derived organoids after transfection was quantified and plotted. ( E ) 5637 was transiently transfected with siRNAs and used to generate organoids. The organoids were subsequently treated with 2.5 µM of CHIR. Images show representative 5637-derived organoids after transfection and treatment at day 1 and 5. Scale bar, 100 µm. ( F ) Relative growth of 5637-derived organoids after transfection and treatment was quantified and plotted. Each dot represents relative growth of one organoid. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001.

    Journal: Oncotarget

    Article Title: Three-dimensional organoid culture reveals involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells

    doi: 10.18632/oncotarget.24308

    Figure Lengend Snippet: β-catenin is required for growth of bladder cancer organoids ( A , D ) Western blot of β-catenin in RT4 and 5637 transiently transfected with siRNAs against β-catenin. Protein was extracted 48 hours after transfection. GAPDH was used as a loading control. NC, negative control. ( B ) RT4 was transiently transfected with siRNAs and used to generate organoids. Images show representative RT4-derived organoids after transfection at day 1 and 6. Scale bar, 100 µm. ( C ) Relative growth of RT4-derived organoids after transfection was quantified and plotted. ( E ) 5637 was transiently transfected with siRNAs and used to generate organoids. The organoids were subsequently treated with 2.5 µM of CHIR. Images show representative 5637-derived organoids after transfection and treatment at day 1 and 5. Scale bar, 100 µm. ( F ) Relative growth of 5637-derived organoids after transfection and treatment was quantified and plotted. Each dot represents relative growth of one organoid. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001.

    Article Snippet: Organoids were incubated at 4° C with anti-β-catenin antibody (610154, 1:500, Becton Dickinson) overnight, then with Alexa-488 conjugated secondary antibody (Thermo Fischer Scientific) for 1 hour.

    Techniques: Western Blot, Transfection, Negative Control, Derivative Assay

    Periostin suppressed the osteogenic differentiation of suture-derived cells via inactivation of Wnt/β-catenin signaling. a The mRNA expression of Wnt-7b, Wnt-1, Wnt-3a, and β-catenin of suture-derived cells after treatment with different concentrations of recombinant mouse periostin. b Western blotting analysis of Wnt/β-catenin signaling markers. c Quantification of Western blotting results. *P

    Journal: Journal of Translational Medicine

    Article Title: Recombinant mouse periostin ameliorates coronal sutures fusion in Twist1+/− mice

    doi: 10.1186/s12967-018-1454-2

    Figure Lengend Snippet: Periostin suppressed the osteogenic differentiation of suture-derived cells via inactivation of Wnt/β-catenin signaling. a The mRNA expression of Wnt-7b, Wnt-1, Wnt-3a, and β-catenin of suture-derived cells after treatment with different concentrations of recombinant mouse periostin. b Western blotting analysis of Wnt/β-catenin signaling markers. c Quantification of Western blotting results. *P

    Article Snippet: The primary antibodies against alkaline phosphatase (ALP), bone sialoprotein (BSP), COL-1, osteocalcin (OCN), runt-related transcription factor 2 (RUNX-2), Wnt-7b, Wnt-3a, Wnt-1, and β-catenin were all purchased from Invitrogen (Carlsbad, CA, USA).

    Techniques: Derivative Assay, Expressing, Recombinant, Western Blot

    IpaC Stabilizes the β-Catenin-Cadherin Interaction (A–C) Infection of Caco-2 cells with S. flexneri -producing WT IpaC or IpaC R362W. (A) Representative images. Five to ten images were analyzed per condition per experiment. Cyan, S. flexneri ; green, membrane-bound YFP; red, β-catenin; blue, DNA. Scale bar 20 βM. (B and C) Quantification of the percentage of protrusions that colocalized with β-catenin in either sub-confluent (B) or confluent (C) cells. Student’s t test. Data are mean ± SEM. (D) Subcellular localization of β-catenin in HeLa cells infected with S. flexneri -producing WT IpaC or IpaC R362W. (E–G) Yeast-based protein interaction assay comparing the efficiency of the interaction between β-catenin and E-cadherin in the presence of either WT IpaC or IpaC R362W. (E) Schematic showing prey is E-cadherin GFP, modifier is IpaC, and bait is β-catenin-μNS. (F) Representative images. Dotted lines are outlines of yeast. Scale bar, 10 μm. A total of 29–175 yeast were analyzed per condition per experiment. (G) Percentage of yeast displaying puncta, which indicates an interaction, from (F). Data are mean ± SEM. N.S., not significant. *p

    Journal: Cell reports

    Article Title: Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread

    doi: 10.1016/j.celrep.2020.108409

    Figure Lengend Snippet: IpaC Stabilizes the β-Catenin-Cadherin Interaction (A–C) Infection of Caco-2 cells with S. flexneri -producing WT IpaC or IpaC R362W. (A) Representative images. Five to ten images were analyzed per condition per experiment. Cyan, S. flexneri ; green, membrane-bound YFP; red, β-catenin; blue, DNA. Scale bar 20 βM. (B and C) Quantification of the percentage of protrusions that colocalized with β-catenin in either sub-confluent (B) or confluent (C) cells. Student’s t test. Data are mean ± SEM. (D) Subcellular localization of β-catenin in HeLa cells infected with S. flexneri -producing WT IpaC or IpaC R362W. (E–G) Yeast-based protein interaction assay comparing the efficiency of the interaction between β-catenin and E-cadherin in the presence of either WT IpaC or IpaC R362W. (E) Schematic showing prey is E-cadherin GFP, modifier is IpaC, and bait is β-catenin-μNS. (F) Representative images. Dotted lines are outlines of yeast. Scale bar, 10 μm. A total of 29–175 yeast were analyzed per condition per experiment. (G) Percentage of yeast displaying puncta, which indicates an interaction, from (F). Data are mean ± SEM. N.S., not significant. *p

    Article Snippet: Western BlotsWestern blots were performed using 1:10,000 mouse anti-β-catenin (BD Biosciences, 610153), 1:1000 mouse anti-vinculin (Sigma, SAB4200729), or 1:1000 rat anti-N-cadherin [Developmental Studies Hybridoma Bank, MNCD2, ( )], 1:1000 rat anti-E-cadherin (Thermo, 14-3249-82) incubating overnight at 4°C, or 1:500 rabbit anti-α-catenin (Thermo, 71-1200), 1:1000 rabbit anti-caveolin 1 (Sigma, C4490), 1:1,000,000 rabbit anti-GroEL (Sigma, G6532), 1:1000 mouse anti-GAPDH (Developmental Studies Hybridoma Bank, DSHB-hGAPDH-2G7), 1:40,000 rabbit anti-keratin 8 (Abcam, Ab53280), 1:10,000 rabbit anti-keratin 18 (Abcam, ab668) 1:5000 goat anti-rabbit HRP (JacksonImmuno, 111-035-144), 1:5000 goat anti-rat HRP (JacksonImmuno, 112-035-003), and 1:5000 goat anti-mouse HRP (JacksonImmuno, 115035003) for 2 hours at room temperature.

    Techniques: Infection, Protein Interaction Assay

    IpaC Interactions with β-Catenin Are Associated with S. flexneri Intercellular Spread (A) Schematic of yeast protein-protein interaction platform. Interaction of the mCherry-tagged prey protein (red) with the bait protein fused to the inclusion body forming protein μNS (gray) results in puncta of red fluorescence. In contrast, the lack of an interaction between prey and bait proteins results in generally diffuse mCherry fluorescence throughout the cytosol of the yeast cells. (B) Protein interaction assay. Arrowheads, fluorescent puncta. Representative images. A total of 88–186 yeast were analyzed per condition per experiment. Scale bar, 10 μM. (C) Percentage of yeast displaying puncta, which indicates an interaction; mean ± SEM. (D and E) Infection of HeLa cells with or without β-catenin knockdown by S. flexneri Δ ipaC -producing WT IpaC or IpaC R362W. (D) Bacterial plaques formed in monolayers stably expressing β-catenin-targeting (#1 and #2) or control shRNA. Images collected at 18 h of infection. Green, S. flexneri ; blue, DNA. Scale bar, 100 μm. Representative images. Five to ten fields were examined per condition per experiment. (E) Quantification of plaque size (area of spread) from (D), mean ± SEM. Dots represent data from three or more independent experiments (C and E). *p

    Journal: Cell reports

    Article Title: Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread

    doi: 10.1016/j.celrep.2020.108409

    Figure Lengend Snippet: IpaC Interactions with β-Catenin Are Associated with S. flexneri Intercellular Spread (A) Schematic of yeast protein-protein interaction platform. Interaction of the mCherry-tagged prey protein (red) with the bait protein fused to the inclusion body forming protein μNS (gray) results in puncta of red fluorescence. In contrast, the lack of an interaction between prey and bait proteins results in generally diffuse mCherry fluorescence throughout the cytosol of the yeast cells. (B) Protein interaction assay. Arrowheads, fluorescent puncta. Representative images. A total of 88–186 yeast were analyzed per condition per experiment. Scale bar, 10 μM. (C) Percentage of yeast displaying puncta, which indicates an interaction; mean ± SEM. (D and E) Infection of HeLa cells with or without β-catenin knockdown by S. flexneri Δ ipaC -producing WT IpaC or IpaC R362W. (D) Bacterial plaques formed in monolayers stably expressing β-catenin-targeting (#1 and #2) or control shRNA. Images collected at 18 h of infection. Green, S. flexneri ; blue, DNA. Scale bar, 100 μm. Representative images. Five to ten fields were examined per condition per experiment. (E) Quantification of plaque size (area of spread) from (D), mean ± SEM. Dots represent data from three or more independent experiments (C and E). *p

    Article Snippet: Western BlotsWestern blots were performed using 1:10,000 mouse anti-β-catenin (BD Biosciences, 610153), 1:1000 mouse anti-vinculin (Sigma, SAB4200729), or 1:1000 rat anti-N-cadherin [Developmental Studies Hybridoma Bank, MNCD2, ( )], 1:1000 rat anti-E-cadherin (Thermo, 14-3249-82) incubating overnight at 4°C, or 1:500 rabbit anti-α-catenin (Thermo, 71-1200), 1:1000 rabbit anti-caveolin 1 (Sigma, C4490), 1:1,000,000 rabbit anti-GroEL (Sigma, G6532), 1:1000 mouse anti-GAPDH (Developmental Studies Hybridoma Bank, DSHB-hGAPDH-2G7), 1:40,000 rabbit anti-keratin 8 (Abcam, Ab53280), 1:10,000 rabbit anti-keratin 18 (Abcam, ab668) 1:5000 goat anti-rabbit HRP (JacksonImmuno, 111-035-144), 1:5000 goat anti-rat HRP (JacksonImmuno, 112-035-003), and 1:5000 goat anti-mouse HRP (JacksonImmuno, 115035003) for 2 hours at room temperature.

    Techniques: Fluorescence, Protein Interaction Assay, Infection, Stable Transfection, Expressing, shRNA

    Effects of Wnt modulation and Fluoxetine treatment on pain thresholds. (A,B) Effects of ablation of β-catenin in hippocampal neural progenitors on mechanical and thermal withdraw thresholds of SNI treated mice. (C,D) Effects of expressing stabilized β-catenin in hippocampal neural progenitors on mechanic and thermal withdraw thresholds of SNI treated mice. (E,F) Effects of Fluoxetine treatment on mechanic and thermal withdraw thresholds of sham injured and SNI treated mice. S, saline. F, Fluoxetine. Notice the no change of pain thresholds in mice with β-catenin manipulation or Fluoxetine treatment. Values represent mean ± SE. One-way ANOVA analysis with Dunnett’s post hoc test was performed in (A–F) . * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Anxiety Specific Response and Contribution of Active Hippocampal Neural Stem Cells to Chronic Pain Through Wnt/β-Catenin Signaling in Mice

    doi: 10.3389/fnmol.2018.00296

    Figure Lengend Snippet: Effects of Wnt modulation and Fluoxetine treatment on pain thresholds. (A,B) Effects of ablation of β-catenin in hippocampal neural progenitors on mechanical and thermal withdraw thresholds of SNI treated mice. (C,D) Effects of expressing stabilized β-catenin in hippocampal neural progenitors on mechanic and thermal withdraw thresholds of SNI treated mice. (E,F) Effects of Fluoxetine treatment on mechanic and thermal withdraw thresholds of sham injured and SNI treated mice. S, saline. F, Fluoxetine. Notice the no change of pain thresholds in mice with β-catenin manipulation or Fluoxetine treatment. Values represent mean ± SE. One-way ANOVA analysis with Dunnett’s post hoc test was performed in (A–F) . * P

    Article Snippet: For immunostaining, the sections were blocked by 0.01 M phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 3% bovine serum albumin (BSA) for 1 h. Primary antibodies were used as following: guinea pig anti-DCX (1:500, Millipore), rat anti-BrdU (1:200, Abcam), rabbit anti-NeuN (1:500, TEMECULA), goat anti-Nestin (1:500, Santa Cruz), rabbit anti-β-gal (1:500, MP), anti-β-catenin (1:200, Millipore).

    Techniques: Mouse Assay, Expressing

    Effects of chronic pain on Wnt activity in ventral hippocampus. (A) Double-immunostaining of Nestin/β-gal and Western-blotting of β-gal in the ventral hippocampus of sham-injured and SNI-treated Topgal mice at 21 dpi. (B) Western-blotting of β-catenin and Axin2 in the ventral hippocampus of sham-injured and SNI-treated mice at 21 dpi. (C) Double-immunostaining and quantification of Nestin/β-gal in the SVZ of sham-injured and SNI-treated Topgal mice at 21 dpi. Inserts in (A) are magnified typical Nestin/β-gal-positive cells in each group, which were pointed by arrows. Bars = 25 μm in (A) and 50 μm in (C) . Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (B,C) . * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Anxiety Specific Response and Contribution of Active Hippocampal Neural Stem Cells to Chronic Pain Through Wnt/β-Catenin Signaling in Mice

    doi: 10.3389/fnmol.2018.00296

    Figure Lengend Snippet: Effects of chronic pain on Wnt activity in ventral hippocampus. (A) Double-immunostaining of Nestin/β-gal and Western-blotting of β-gal in the ventral hippocampus of sham-injured and SNI-treated Topgal mice at 21 dpi. (B) Western-blotting of β-catenin and Axin2 in the ventral hippocampus of sham-injured and SNI-treated mice at 21 dpi. (C) Double-immunostaining and quantification of Nestin/β-gal in the SVZ of sham-injured and SNI-treated Topgal mice at 21 dpi. Inserts in (A) are magnified typical Nestin/β-gal-positive cells in each group, which were pointed by arrows. Bars = 25 μm in (A) and 50 μm in (C) . Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (B,C) . * P

    Article Snippet: For immunostaining, the sections were blocked by 0.01 M phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 3% bovine serum albumin (BSA) for 1 h. Primary antibodies were used as following: guinea pig anti-DCX (1:500, Millipore), rat anti-BrdU (1:200, Abcam), rabbit anti-NeuN (1:500, TEMECULA), goat anti-Nestin (1:500, Santa Cruz), rabbit anti-β-gal (1:500, MP), anti-β-catenin (1:200, Millipore).

    Techniques: Activity Assay, Double Immunostaining, Western Blot, Mouse Assay, Two Tailed Test

    Effects of expressing stabilized β-catenin in adult neural progenitors on hippocampal neurogenesis and pain-associated anxiety. (A) Schematic drawing of the experimental design. (B) Double-immunostaining of Nestin/β-catenin and quantification of Nestin-positive cells SNI-treated wild-type (WT) or Nestin-β-catEX3 mice. Notice the enhanced expression of β-catenin and the increase of horizontal Nestin-positive cells in the hippocampus of Nestin-β-catEX3 mice. (C) Double-immunostaining and quantification of BrdU/DCX in SNI-treated WT or Nestin-β-catEX3 mice. (D,E) Open field and elevated plus maze tests of SNI-treated WT or Nestin-β-catEX3 mice at 21 dpi. Notice the alleviation of anxiety in Nestin-β-catEX3 mice with chronic pain. OA, open arm. Inserts in (B,C) are magnified typical double-stained cells in each group, which were pointed by arrows. Bars = 25 μm in (B) and 50 μm in (C) . Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (B,C) . * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Anxiety Specific Response and Contribution of Active Hippocampal Neural Stem Cells to Chronic Pain Through Wnt/β-Catenin Signaling in Mice

    doi: 10.3389/fnmol.2018.00296

    Figure Lengend Snippet: Effects of expressing stabilized β-catenin in adult neural progenitors on hippocampal neurogenesis and pain-associated anxiety. (A) Schematic drawing of the experimental design. (B) Double-immunostaining of Nestin/β-catenin and quantification of Nestin-positive cells SNI-treated wild-type (WT) or Nestin-β-catEX3 mice. Notice the enhanced expression of β-catenin and the increase of horizontal Nestin-positive cells in the hippocampus of Nestin-β-catEX3 mice. (C) Double-immunostaining and quantification of BrdU/DCX in SNI-treated WT or Nestin-β-catEX3 mice. (D,E) Open field and elevated plus maze tests of SNI-treated WT or Nestin-β-catEX3 mice at 21 dpi. Notice the alleviation of anxiety in Nestin-β-catEX3 mice with chronic pain. OA, open arm. Inserts in (B,C) are magnified typical double-stained cells in each group, which were pointed by arrows. Bars = 25 μm in (B) and 50 μm in (C) . Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (B,C) . * P

    Article Snippet: For immunostaining, the sections were blocked by 0.01 M phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 3% bovine serum albumin (BSA) for 1 h. Primary antibodies were used as following: guinea pig anti-DCX (1:500, Millipore), rat anti-BrdU (1:200, Abcam), rabbit anti-NeuN (1:500, TEMECULA), goat anti-Nestin (1:500, Santa Cruz), rabbit anti-β-gal (1:500, MP), anti-β-catenin (1:200, Millipore).

    Techniques: Expressing, Double Immunostaining, Mouse Assay, Staining, Two Tailed Test

    Effects of ablating β-catenin in adult neural progenitors on hippocampal neurogenesis and pain-associated anxiety. (A) Schematic drawing of the experimental design. (B) Double-immunostaining of Nestin/β-catenin and quantification of Nestin-positive cells in SNI-treated WT or Nestin-β-cat CKO mice at 10 dpi. Notice the decrease of β-catenin immunoreactivity and radial Nestin-positive cells in the hippocampus of Nestin-β-cat CKO mice. (C) Double-immunostaining and quantification of BrdU/DCX in SNI-treated WT or Nestin-β-cat CKO mice at 10 dpi. (D,E) Open field and elevated plus maze tests of SNI-treated WT or Nestin-β-cat CKO mice at 10 dpi. Notice the appearance of anxiety in Nestin-β-cat CKO mice at 10 dpi. OA, open arm. Inserts in (B,C) are magnified typical double-stained cells in each group, which were pointed by arrows. Bars = 25 μm in (B) and 50 μm in (C) . Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (B–E) . * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Anxiety Specific Response and Contribution of Active Hippocampal Neural Stem Cells to Chronic Pain Through Wnt/β-Catenin Signaling in Mice

    doi: 10.3389/fnmol.2018.00296

    Figure Lengend Snippet: Effects of ablating β-catenin in adult neural progenitors on hippocampal neurogenesis and pain-associated anxiety. (A) Schematic drawing of the experimental design. (B) Double-immunostaining of Nestin/β-catenin and quantification of Nestin-positive cells in SNI-treated WT or Nestin-β-cat CKO mice at 10 dpi. Notice the decrease of β-catenin immunoreactivity and radial Nestin-positive cells in the hippocampus of Nestin-β-cat CKO mice. (C) Double-immunostaining and quantification of BrdU/DCX in SNI-treated WT or Nestin-β-cat CKO mice at 10 dpi. (D,E) Open field and elevated plus maze tests of SNI-treated WT or Nestin-β-cat CKO mice at 10 dpi. Notice the appearance of anxiety in Nestin-β-cat CKO mice at 10 dpi. OA, open arm. Inserts in (B,C) are magnified typical double-stained cells in each group, which were pointed by arrows. Bars = 25 μm in (B) and 50 μm in (C) . Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (B–E) . * P

    Article Snippet: For immunostaining, the sections were blocked by 0.01 M phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 3% bovine serum albumin (BSA) for 1 h. Primary antibodies were used as following: guinea pig anti-DCX (1:500, Millipore), rat anti-BrdU (1:200, Abcam), rabbit anti-NeuN (1:500, TEMECULA), goat anti-Nestin (1:500, Santa Cruz), rabbit anti-β-gal (1:500, MP), anti-β-catenin (1:200, Millipore).

    Techniques: Double Immunostaining, Mouse Assay, Staining, Two Tailed Test

    Effects of Fluoxetine treatment on the Wnt activity in hippocampus. (A) Double-immunostaining of Nestin/β-gal in sham mice treated with saline (Sham+S), sham mice treated with Fluoxetine (Sham+F), SNI mice treated with saline (SNI+S), and SNI mice treated with Fluoxetine (SNI+F) at 21 dpi. Notice the significant increase of radial Nestin-positive cells in Fluoxetine treated mice. (B) Western-blotting of β-gal and β-catenin in Topgal mice with the following treatments: sham injury plus saline (Sham+S), sham injury plus Fluoxetine (Sham+F), SNI plus saline (SNI+S) and SNI plus Fluoxetine (SNI+F). Notice that Fluoxetine treatment significantly increased the expression level of β-gal and β-catenin in both sham and SNI-treated mice, as compared to corresponding saline controls. S, saline. F, Fluoxetine. Inserts in (A) are typical double-stained cells in each group, which were pointed by arrows. Bars = 50 μm. Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (A,B) . * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Anxiety Specific Response and Contribution of Active Hippocampal Neural Stem Cells to Chronic Pain Through Wnt/β-Catenin Signaling in Mice

    doi: 10.3389/fnmol.2018.00296

    Figure Lengend Snippet: Effects of Fluoxetine treatment on the Wnt activity in hippocampus. (A) Double-immunostaining of Nestin/β-gal in sham mice treated with saline (Sham+S), sham mice treated with Fluoxetine (Sham+F), SNI mice treated with saline (SNI+S), and SNI mice treated with Fluoxetine (SNI+F) at 21 dpi. Notice the significant increase of radial Nestin-positive cells in Fluoxetine treated mice. (B) Western-blotting of β-gal and β-catenin in Topgal mice with the following treatments: sham injury plus saline (Sham+S), sham injury plus Fluoxetine (Sham+F), SNI plus saline (SNI+S) and SNI plus Fluoxetine (SNI+F). Notice that Fluoxetine treatment significantly increased the expression level of β-gal and β-catenin in both sham and SNI-treated mice, as compared to corresponding saline controls. S, saline. F, Fluoxetine. Inserts in (A) are typical double-stained cells in each group, which were pointed by arrows. Bars = 50 μm. Values represent mean ± SE. Unpaired, two tailed Student’s t -tests were performed in (A,B) . * P

    Article Snippet: For immunostaining, the sections were blocked by 0.01 M phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 3% bovine serum albumin (BSA) for 1 h. Primary antibodies were used as following: guinea pig anti-DCX (1:500, Millipore), rat anti-BrdU (1:200, Abcam), rabbit anti-NeuN (1:500, TEMECULA), goat anti-Nestin (1:500, Santa Cruz), rabbit anti-β-gal (1:500, MP), anti-β-catenin (1:200, Millipore).

    Techniques: Activity Assay, Double Immunostaining, Mouse Assay, Western Blot, Expressing, Staining, Two Tailed Test

    The increased mRNA and protein levels of β-catenin were partially reversed by addition of a Wnt/β-catenin signaling inhibitor (DKK1). ( a ) The β-catenin mRNA levels in control, morroniside and morronside +DKK1 groups were determined by RT-PCR after treatment for 24 h. ( b ) Total β-catenin protein levels in control, morroniside and morronside +DKK1 groups were determined by western blotting. The grouping of blots was cropped from the same gel. ( c ) Quantitative analysis of β-catenin protein levels in control, morroniside and morroniside +DKK1 groups. a.u., arbitrary units.

    Journal: Scientific Reports

    Article Title: Morroniside regulates hair growth and cycle transition via activation of the Wnt/β-catenin signaling pathway

    doi: 10.1038/s41598-018-32138-2

    Figure Lengend Snippet: The increased mRNA and protein levels of β-catenin were partially reversed by addition of a Wnt/β-catenin signaling inhibitor (DKK1). ( a ) The β-catenin mRNA levels in control, morroniside and morronside +DKK1 groups were determined by RT-PCR after treatment for 24 h. ( b ) Total β-catenin protein levels in control, morroniside and morronside +DKK1 groups were determined by western blotting. The grouping of blots was cropped from the same gel. ( c ) Quantitative analysis of β-catenin protein levels in control, morroniside and morroniside +DKK1 groups. a.u., arbitrary units.

    Article Snippet: The primary antibody used in this study was rabbit anti-β-catenin (1:250; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

    Morroniside activated the Wnt/β-catenin signaling pathway. ( a – c ) Relative mRNA expression levels of Wnt10b, β-catenin, and lef1 in human ORSCs, determined by RT-PCR. ( d ) Wnt10b, β-catenin (total, cytoplasmic and nuclear), and lef1 protein expression levels in human ORSCs, detected by western blotting. The grouping of blots was cropped from different gels. GAPDH was used as the control for total and cytoplasmic proteins, whereas histone 2A was used as the control for the nuclear proteins. ( e – i ) Quantitative analysis of Wnt10b, β-catenin (total, cytoplasmic and nuclear), and lef1 protein levels. ( j ) TOPFlash reporter activity induction in human ORSCs was detected by TOPFlash reporter assay. a.u., arbitrary units. ( k ) β-catenin is shown in red, and nuclei counterstained with DAPI (blue). Merged images indicate the expression and location of β-catenin. *P

    Journal: Scientific Reports

    Article Title: Morroniside regulates hair growth and cycle transition via activation of the Wnt/β-catenin signaling pathway

    doi: 10.1038/s41598-018-32138-2

    Figure Lengend Snippet: Morroniside activated the Wnt/β-catenin signaling pathway. ( a – c ) Relative mRNA expression levels of Wnt10b, β-catenin, and lef1 in human ORSCs, determined by RT-PCR. ( d ) Wnt10b, β-catenin (total, cytoplasmic and nuclear), and lef1 protein expression levels in human ORSCs, detected by western blotting. The grouping of blots was cropped from different gels. GAPDH was used as the control for total and cytoplasmic proteins, whereas histone 2A was used as the control for the nuclear proteins. ( e – i ) Quantitative analysis of Wnt10b, β-catenin (total, cytoplasmic and nuclear), and lef1 protein levels. ( j ) TOPFlash reporter activity induction in human ORSCs was detected by TOPFlash reporter assay. a.u., arbitrary units. ( k ) β-catenin is shown in red, and nuclei counterstained with DAPI (blue). Merged images indicate the expression and location of β-catenin. *P

    Article Snippet: The primary antibody used in this study was rabbit anti-β-catenin (1:250; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Reporter Assay

    The increased proliferation and migration caused by morroniside were partially rescued by addition of a Wnt/β-catenin signaling inhibitor (DKK1). ( a) The proliferation of ORSCs in control, morroniside and morronside +DKK1 groups was measured by MTS assays at 0, 6, 12, 24, 48, and 72 h, respectively. ( b , c ) Distributions of ORSCs in G1, S, and G2 phases in control, morroniside and morronside +DKK1 groups were detected by flow cytometry. ( d ) Fold changes in the fractions of cells in S and S/G1 phases in control, morroniside and morronside +DKK1 groups. ( e , f ) Transwell assays of ORSCs in control, morroniside and morronside +DKK1 groups. ( g , h ) Wound scratch assays of ORSCs in control, morroniside and morronside +DKK1 groups. a.u., arbitrary units.

    Journal: Scientific Reports

    Article Title: Morroniside regulates hair growth and cycle transition via activation of the Wnt/β-catenin signaling pathway

    doi: 10.1038/s41598-018-32138-2

    Figure Lengend Snippet: The increased proliferation and migration caused by morroniside were partially rescued by addition of a Wnt/β-catenin signaling inhibitor (DKK1). ( a) The proliferation of ORSCs in control, morroniside and morronside +DKK1 groups was measured by MTS assays at 0, 6, 12, 24, 48, and 72 h, respectively. ( b , c ) Distributions of ORSCs in G1, S, and G2 phases in control, morroniside and morronside +DKK1 groups were detected by flow cytometry. ( d ) Fold changes in the fractions of cells in S and S/G1 phases in control, morroniside and morronside +DKK1 groups. ( e , f ) Transwell assays of ORSCs in control, morroniside and morronside +DKK1 groups. ( g , h ) Wound scratch assays of ORSCs in control, morroniside and morronside +DKK1 groups. a.u., arbitrary units.

    Article Snippet: The primary antibody used in this study was rabbit anti-β-catenin (1:250; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, Flow Cytometry, Cytometry

    Gene expression during neural crest induction in the zebrafish embryo and upon Wnt stimulation in SKMEL28 melanoma cells, and clinical significance. a Heatmaps of real-time qPCR gene expression analysis of the melanoma cell lines SKMEL19, 451 LU, BLM and A375 for AXIN2. TYR, MITF and the four genes INHBA, CYR61, ANGTPL4 and FABP7. Log2 transformed x-fold expression values were used for color-coding. Yellow: upregulated gene expression; blue: downregulated gene expression at 24 h treatment of the melanoma cells; black: not detectable b Western blot analysis to detect protein levels of beta-catenin, PTEN, phospo-Ser473 (AKT) and AKT in the melanoma cell lines A375, SKMEL19, BLM and 451 LU after treatment with 3T3-CM, 3T3-Wnt3a, or 0.5 μM PKF115–584 for 24 h. Beta-actin was used as loading control. Small-hairpin knockdown cells served as samples to investigate the dependence on beta-catenin. c Luciferase reporter assay (Super8xTOPFlash) indicates a 3-fold activation of the canonical Wnt−/β-catenin signaling pathway after stimulation of SKMEL28 cells with Wnt3a-conditioned medium (3T3-Wnt3a). PKF115–584 treatment (0.5 μM for 12 h) significantly inhibited reporter activity. **: p

    Journal: Molecular Cancer

    Article Title: Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype

    doi: 10.1186/s12943-018-0773-5

    Figure Lengend Snippet: Gene expression during neural crest induction in the zebrafish embryo and upon Wnt stimulation in SKMEL28 melanoma cells, and clinical significance. a Heatmaps of real-time qPCR gene expression analysis of the melanoma cell lines SKMEL19, 451 LU, BLM and A375 for AXIN2. TYR, MITF and the four genes INHBA, CYR61, ANGTPL4 and FABP7. Log2 transformed x-fold expression values were used for color-coding. Yellow: upregulated gene expression; blue: downregulated gene expression at 24 h treatment of the melanoma cells; black: not detectable b Western blot analysis to detect protein levels of beta-catenin, PTEN, phospo-Ser473 (AKT) and AKT in the melanoma cell lines A375, SKMEL19, BLM and 451 LU after treatment with 3T3-CM, 3T3-Wnt3a, or 0.5 μM PKF115–584 for 24 h. Beta-actin was used as loading control. Small-hairpin knockdown cells served as samples to investigate the dependence on beta-catenin. c Luciferase reporter assay (Super8xTOPFlash) indicates a 3-fold activation of the canonical Wnt−/β-catenin signaling pathway after stimulation of SKMEL28 cells with Wnt3a-conditioned medium (3T3-Wnt3a). PKF115–584 treatment (0.5 μM for 12 h) significantly inhibited reporter activity. **: p

    Article Snippet: Immunohistochemistry of a human melanoma tissue microarray (TMA) using an anti-β-catenin antibody (1:100, Cell Signaling #9562) was performed as described previously [ ].

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Western Blot, Luciferase, Reporter Assay, Activation Assay, Activity Assay

    Knock-down of β-catenin with shRNA in SKMEL28 cells inhibits brain metastasis formation in the rhombencephalon of the chick embryo. a SKMEL28 aggregates were embedded into a collagen matrix with or without 3T3 or 3T3-Wnt3a stroma cells. Sprouting of SKMEL28 cells was measured at 0 h and 48 h, and an invasion index calculated as ratio of the 48 h to the 0 h value. 3T3 stroma cells, and more prominently, 3T3-Wnt3a stroma cells increased sprouting of SKMEL28 cells (**: p

    Journal: Molecular Cancer

    Article Title: Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype

    doi: 10.1186/s12943-018-0773-5

    Figure Lengend Snippet: Knock-down of β-catenin with shRNA in SKMEL28 cells inhibits brain metastasis formation in the rhombencephalon of the chick embryo. a SKMEL28 aggregates were embedded into a collagen matrix with or without 3T3 or 3T3-Wnt3a stroma cells. Sprouting of SKMEL28 cells was measured at 0 h and 48 h, and an invasion index calculated as ratio of the 48 h to the 0 h value. 3T3 stroma cells, and more prominently, 3T3-Wnt3a stroma cells increased sprouting of SKMEL28 cells (**: p

    Article Snippet: Immunohistochemistry of a human melanoma tissue microarray (TMA) using an anti-β-catenin antibody (1:100, Cell Signaling #9562) was performed as described previously [ ].

    Techniques: shRNA

    Expression of β-catenin in BLM melanoma cells during brain metastasis in vivo . Untreated ( n = 8) or 0.5 μM PKF115–584 pre-conditioned ( n = 8) human BLM melanoma cell aggregates were injected into the IV th ventricle (rhombencephalon) of the stage 12/13 HH chick embryo ( m ). After 96 h of further incubation the embryos were analyzed for tumor formation and melanoma cell invasion. a Untreated BLM cells had formed small tumor nodules in the roof plate of the rhombencephalon and infiltrated the neighboring host tissues in 7/8 chick embryos. b The HMB45-negative BLM cells the neural crest marker p75 (red cytoplasmic staining). c-e : BLM cells were almost 100% MIB1-positive (red nuclear staining) and were detected close to ( d ) or within ( e , arrows pointing at BLM cells) blood vessels. f-i Immunohistochemistry for anti-β-catenin ( f , red staining) showed that in the central tumor nodule as well as the metastatic nodule in the region of the floor plate the MIB1-positive BLM cells with a compact, epithelial-like morphology (asterisks in g and h ) had only a weak cytoplasmic expression of β-catenin, while the cells in the outskirt of the tumor nodule and cells invading the host tissues in the mesenchyme along the neuroepithelium with a stretched, mesenchymal-like morphology (arrows in g and i ) showed a prominent β-catenin expression. j-l In 8/8 embryos injected with PKF115–584 pre-conditioned BLM cells only an empty space as reminder of the former tumor nodule was detected after 96 h ( j, k ). Only few PKF115–584 pre-conditioned, MIB1-negative BLM cells and aggregates with morphological signs of apoptosis could be distinguished at this point in the lumen of the IVth ventricle floating among cellular debris of dead BLM cells ( l ). m Scheme of the injection of melanoma cells into the rhombencephalon of chick embryos. n Qualitative enumeration of chick embryos showing viable BLM melanoma cells in the roof plate with invading melanoma cells as shown in A-L. (asterisks define p -values calculated by Fisher’s exact test)

    Journal: Molecular Cancer

    Article Title: Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype

    doi: 10.1186/s12943-018-0773-5

    Figure Lengend Snippet: Expression of β-catenin in BLM melanoma cells during brain metastasis in vivo . Untreated ( n = 8) or 0.5 μM PKF115–584 pre-conditioned ( n = 8) human BLM melanoma cell aggregates were injected into the IV th ventricle (rhombencephalon) of the stage 12/13 HH chick embryo ( m ). After 96 h of further incubation the embryos were analyzed for tumor formation and melanoma cell invasion. a Untreated BLM cells had formed small tumor nodules in the roof plate of the rhombencephalon and infiltrated the neighboring host tissues in 7/8 chick embryos. b The HMB45-negative BLM cells the neural crest marker p75 (red cytoplasmic staining). c-e : BLM cells were almost 100% MIB1-positive (red nuclear staining) and were detected close to ( d ) or within ( e , arrows pointing at BLM cells) blood vessels. f-i Immunohistochemistry for anti-β-catenin ( f , red staining) showed that in the central tumor nodule as well as the metastatic nodule in the region of the floor plate the MIB1-positive BLM cells with a compact, epithelial-like morphology (asterisks in g and h ) had only a weak cytoplasmic expression of β-catenin, while the cells in the outskirt of the tumor nodule and cells invading the host tissues in the mesenchyme along the neuroepithelium with a stretched, mesenchymal-like morphology (arrows in g and i ) showed a prominent β-catenin expression. j-l In 8/8 embryos injected with PKF115–584 pre-conditioned BLM cells only an empty space as reminder of the former tumor nodule was detected after 96 h ( j, k ). Only few PKF115–584 pre-conditioned, MIB1-negative BLM cells and aggregates with morphological signs of apoptosis could be distinguished at this point in the lumen of the IVth ventricle floating among cellular debris of dead BLM cells ( l ). m Scheme of the injection of melanoma cells into the rhombencephalon of chick embryos. n Qualitative enumeration of chick embryos showing viable BLM melanoma cells in the roof plate with invading melanoma cells as shown in A-L. (asterisks define p -values calculated by Fisher’s exact test)

    Article Snippet: Immunohistochemistry of a human melanoma tissue microarray (TMA) using an anti-β-catenin antibody (1:100, Cell Signaling #9562) was performed as described previously [ ].

    Techniques: Expressing, In Vivo, Injection, Incubation, Marker, Staining, Immunohistochemistry

    β-catenin is expressed in primary human melanomas in particular in cells of the invasive front. a Cytoplasmic expression of β-catenin in primary melanomas. A stronger expression of β-catenin in melanoma cells of the invasive front was observed by immunohistochemistry when compared to the bulk cells of the primary melanoma. Melanoma cells of the invasive front with a high expression of β-catenin had a spindle-like, mesenchymal morphology. Overview (upper row) and higher magnification of the invasive front (lower row) of four representative primary melanomas. b Overall survival (OS) analysis of the TCGA data set for the top and bottom ( n = 50 each) WNT3A and WNT5A expressing melanomas reveal a significantly worse OS for WNT3A-high ( p = 0.03) but not WNT5A-high melanoma patients ( p = 0.39)

    Journal: Molecular Cancer

    Article Title: Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype

    doi: 10.1186/s12943-018-0773-5

    Figure Lengend Snippet: β-catenin is expressed in primary human melanomas in particular in cells of the invasive front. a Cytoplasmic expression of β-catenin in primary melanomas. A stronger expression of β-catenin in melanoma cells of the invasive front was observed by immunohistochemistry when compared to the bulk cells of the primary melanoma. Melanoma cells of the invasive front with a high expression of β-catenin had a spindle-like, mesenchymal morphology. Overview (upper row) and higher magnification of the invasive front (lower row) of four representative primary melanomas. b Overall survival (OS) analysis of the TCGA data set for the top and bottom ( n = 50 each) WNT3A and WNT5A expressing melanomas reveal a significantly worse OS for WNT3A-high ( p = 0.03) but not WNT5A-high melanoma patients ( p = 0.39)

    Article Snippet: Immunohistochemistry of a human melanoma tissue microarray (TMA) using an anti-β-catenin antibody (1:100, Cell Signaling #9562) was performed as described previously [ ].

    Techniques: Expressing, Immunohistochemistry

    Effect of LINC00704 knockdown on thyroid cancer cells apoptosis and invasion. A, B, The cell apoptotic rate of BHT101 and BCPAP cells after transfection with LINC00704 siRNAs or negative control was determined using Flow cytometry assays. C, D, Transwell assays were performed to evaluate the invasive ability of si‐LINC00704 or si‐NC transfected BHT101 cells. Data represent the mean ± SD from three independent experiments. E, F, The protein levels of E‐cadherin, N‐cadherin and β‐catenin in si‐LINC00704 or si‐NC transfected BHT101 cells were examined by western blot. ** P

    Journal: Cancer Medicine

    Article Title: Identification of differential expressed lnc RNAs in human thyroid cancer by a genome‐wide analyses. Identification of differential expressed lncRNAs in human thyroid cancer by a genome‐wide analyses

    doi: 10.1002/cam4.1627

    Figure Lengend Snippet: Effect of LINC00704 knockdown on thyroid cancer cells apoptosis and invasion. A, B, The cell apoptotic rate of BHT101 and BCPAP cells after transfection with LINC00704 siRNAs or negative control was determined using Flow cytometry assays. C, D, Transwell assays were performed to evaluate the invasive ability of si‐LINC00704 or si‐NC transfected BHT101 cells. Data represent the mean ± SD from three independent experiments. E, F, The protein levels of E‐cadherin, N‐cadherin and β‐catenin in si‐LINC00704 or si‐NC transfected BHT101 cells were examined by western blot. ** P

    Article Snippet: GAPDH, E‐cadherin, N‐cadherin, Vimentine, and β‐catenin antibodies were purchased from Cell Signaling Technology.

    Techniques: Transfection, Negative Control, Flow Cytometry, Cytometry, Western Blot

    Compared to oral gavage, intraperitoneal 4-AAQB significantly and more efficiently suppresses GBM stem cell-induced tumor growth, in vivo. ( A ) Tumor size vs time curve show the inhibitory effect of 4-AAQB on U87MG tumor growth either via p.o or i.p route, as compared to the control group. ( B ) Photographs of tumor samples harvested from the in vivo studies. ( C ) The differential effects of oral and intraperitoneal administration of 4-AAQB on the expression and localization of TCF-1 and β-catenin in xenograft-derived GBM primary culture. * p

    Journal: Cancers

    Article Title: The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo

    doi: 10.3390/cancers10120491

    Figure Lengend Snippet: Compared to oral gavage, intraperitoneal 4-AAQB significantly and more efficiently suppresses GBM stem cell-induced tumor growth, in vivo. ( A ) Tumor size vs time curve show the inhibitory effect of 4-AAQB on U87MG tumor growth either via p.o or i.p route, as compared to the control group. ( B ) Photographs of tumor samples harvested from the in vivo studies. ( C ) The differential effects of oral and intraperitoneal administration of 4-AAQB on the expression and localization of TCF-1 and β-catenin in xenograft-derived GBM primary culture. * p

    Article Snippet: Followed by incubating the cells with antibody against β-catenin (D10A8; XP Rabbit mAb #8480; Cell Signaling Technology), Sox2 (D6D9; XP Rabbit mAb #3579; Cell Signaling Technology), Oct4 (C30A3; Rabbit mAb #2840; Cell Signaling Technology), or F-actin (13E5; Rabbit mAb #4970; Cell Signaling Technology) diluted 1:500, in PBS containing 3% normal goat serum at room temperature for 2 h. For negative controls we omitted the primary antibody.

    Techniques: In Vivo, Expressing, Derivative Assay

    4-AAQB markedly inhibits the stem cell-like phenotype of U87MG and DBTRG-05MG cells. ( A ) Representative photo-images and ( B ) graphical quantification showing 4-AAQB significantly reduce the size and number of U87MG and DBTRG-05MG primary, secondary, and tertiary generation tumorspheres formed. Original magnification = 200×. ( C ) Representative photo-images and ( D ) graphical representations of 4-AAQB effect on the ability of U87MG and DBTRG-05MG cells to form colonies; ( E ) The dose-dependent inhibitory effect of 5–10 M 4-AAQB treatment on the expression levels of β-catenin, c-Myc, KLF4, Oct4, and Sox2 proteins in DBTRG05MG cells shown in a representative Western blot data. β-actin served as loading control. * p

    Journal: Cancers

    Article Title: The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo

    doi: 10.3390/cancers10120491

    Figure Lengend Snippet: 4-AAQB markedly inhibits the stem cell-like phenotype of U87MG and DBTRG-05MG cells. ( A ) Representative photo-images and ( B ) graphical quantification showing 4-AAQB significantly reduce the size and number of U87MG and DBTRG-05MG primary, secondary, and tertiary generation tumorspheres formed. Original magnification = 200×. ( C ) Representative photo-images and ( D ) graphical representations of 4-AAQB effect on the ability of U87MG and DBTRG-05MG cells to form colonies; ( E ) The dose-dependent inhibitory effect of 5–10 M 4-AAQB treatment on the expression levels of β-catenin, c-Myc, KLF4, Oct4, and Sox2 proteins in DBTRG05MG cells shown in a representative Western blot data. β-actin served as loading control. * p

    Article Snippet: Followed by incubating the cells with antibody against β-catenin (D10A8; XP Rabbit mAb #8480; Cell Signaling Technology), Sox2 (D6D9; XP Rabbit mAb #3579; Cell Signaling Technology), Oct4 (C30A3; Rabbit mAb #2840; Cell Signaling Technology), or F-actin (13E5; Rabbit mAb #4970; Cell Signaling Technology) diluted 1:500, in PBS containing 3% normal goat serum at room temperature for 2 h. For negative controls we omitted the primary antibody.

    Techniques: Expressing, Western Blot

    4-AAQB significantly suppresses the viability and oncogenicity of GBM cells. ( A ) The dose-dependent cytotoxic effect of 4-AAQB on the cell viability of U-87MG and DBTRG-05MG. ( B ) Representative photo-images and graphical quantification showing the effect of 5 μM and 10 μM of 4-AAQB on the migration of U-87MG and DBTRG-05MG over 24 h duration. ( C ) 4-AAQB dose-dependently inhibit the number of invaded U-87MG and DBTRG-05MG cells. Scale bar: 50 μm. ( D ) The inhibitory effect of 5 M and 10 M 4-AAQB on the expression levels of β-catenin, vimentin, and slug proteins in DBTRG-05MG cells as shown by Western blot assay. β-actin served as loading control. * p

    Journal: Cancers

    Article Title: The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo

    doi: 10.3390/cancers10120491

    Figure Lengend Snippet: 4-AAQB significantly suppresses the viability and oncogenicity of GBM cells. ( A ) The dose-dependent cytotoxic effect of 4-AAQB on the cell viability of U-87MG and DBTRG-05MG. ( B ) Representative photo-images and graphical quantification showing the effect of 5 μM and 10 μM of 4-AAQB on the migration of U-87MG and DBTRG-05MG over 24 h duration. ( C ) 4-AAQB dose-dependently inhibit the number of invaded U-87MG and DBTRG-05MG cells. Scale bar: 50 μm. ( D ) The inhibitory effect of 5 M and 10 M 4-AAQB on the expression levels of β-catenin, vimentin, and slug proteins in DBTRG-05MG cells as shown by Western blot assay. β-actin served as loading control. * p

    Article Snippet: Followed by incubating the cells with antibody against β-catenin (D10A8; XP Rabbit mAb #8480; Cell Signaling Technology), Sox2 (D6D9; XP Rabbit mAb #3579; Cell Signaling Technology), Oct4 (C30A3; Rabbit mAb #2840; Cell Signaling Technology), or F-actin (13E5; Rabbit mAb #4970; Cell Signaling Technology) diluted 1:500, in PBS containing 3% normal goat serum at room temperature for 2 h. For negative controls we omitted the primary antibody.

    Techniques: Migration, Expressing, Western Blot

    β-catenin facilitates GBM oncogenicity and disease recurrence, as well as their cancer stem cell-like traits. ( A ) TCGA brain dataset ( n = 557) using the Human Genome U133A Array showing the differential β-catenin mRNA expression in GBM ( n = 547) and normal brain ( n = 10) samples. ( B ) Immunohistochemistry (IHC) staining comparing expression of β-catenin in GBM, recurrent and adjacent non-tumor brain tissues in 2 Taipei Medical University Shuang Ho Hospital (TMU-SHH) brain tumor patients. * p

    Journal: Cancers

    Article Title: The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo

    doi: 10.3390/cancers10120491

    Figure Lengend Snippet: β-catenin facilitates GBM oncogenicity and disease recurrence, as well as their cancer stem cell-like traits. ( A ) TCGA brain dataset ( n = 557) using the Human Genome U133A Array showing the differential β-catenin mRNA expression in GBM ( n = 547) and normal brain ( n = 10) samples. ( B ) Immunohistochemistry (IHC) staining comparing expression of β-catenin in GBM, recurrent and adjacent non-tumor brain tissues in 2 Taipei Medical University Shuang Ho Hospital (TMU-SHH) brain tumor patients. * p

    Article Snippet: Followed by incubating the cells with antibody against β-catenin (D10A8; XP Rabbit mAb #8480; Cell Signaling Technology), Sox2 (D6D9; XP Rabbit mAb #3579; Cell Signaling Technology), Oct4 (C30A3; Rabbit mAb #2840; Cell Signaling Technology), or F-actin (13E5; Rabbit mAb #4970; Cell Signaling Technology) diluted 1:500, in PBS containing 3% normal goat serum at room temperature for 2 h. For negative controls we omitted the primary antibody.

    Techniques: Expressing, Immunohistochemistry, Staining

    4-AAQB disrupts β-catenin/TCF-1/Stat3 signaling axis in GBM cells. ( A ) Chemical structure of 4-Acetylantroquinonol B; C26H38O7, 462.58 g/mol. Representative Western blot data and graphical quantification showing ( B ) the knock-down efficiency of β-catenin in human GBM cell line U87MG, with corresponding effect on the expression levels of pGSK3β, GSK-3β, TCF1/TCF7, and LEF1 proteins; and ( C ) the dose-dependent downregulation of β-catenin, pGSK3β, TCF1/TCF7, LEF1, and p-Stat3 proteins in U87MG and DBTRG05MG cells treated with increasing concentrations of 4-AAQB for 24 h. β-actin served as loading control. siCTNNB1, short interfering RNA directed specifically against β-catenin; WT, wild type; * p

    Journal: Cancers

    Article Title: The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo

    doi: 10.3390/cancers10120491

    Figure Lengend Snippet: 4-AAQB disrupts β-catenin/TCF-1/Stat3 signaling axis in GBM cells. ( A ) Chemical structure of 4-Acetylantroquinonol B; C26H38O7, 462.58 g/mol. Representative Western blot data and graphical quantification showing ( B ) the knock-down efficiency of β-catenin in human GBM cell line U87MG, with corresponding effect on the expression levels of pGSK3β, GSK-3β, TCF1/TCF7, and LEF1 proteins; and ( C ) the dose-dependent downregulation of β-catenin, pGSK3β, TCF1/TCF7, LEF1, and p-Stat3 proteins in U87MG and DBTRG05MG cells treated with increasing concentrations of 4-AAQB for 24 h. β-actin served as loading control. siCTNNB1, short interfering RNA directed specifically against β-catenin; WT, wild type; * p

    Article Snippet: Followed by incubating the cells with antibody against β-catenin (D10A8; XP Rabbit mAb #8480; Cell Signaling Technology), Sox2 (D6D9; XP Rabbit mAb #3579; Cell Signaling Technology), Oct4 (C30A3; Rabbit mAb #2840; Cell Signaling Technology), or F-actin (13E5; Rabbit mAb #4970; Cell Signaling Technology) diluted 1:500, in PBS containing 3% normal goat serum at room temperature for 2 h. For negative controls we omitted the primary antibody.

    Techniques: Western Blot, Expressing, Small Interfering RNA

    4-AAQB inhibits the nuclear localization of β-catenin, Sox2, and Oct4 in GBM cells. U87MG and DBTRG-05MG cells treated with or without 5–10 μM 4-AAQB were immunostained with Sox2 (green), Oct4 (red), β-catenin (red), and F-actin (green) antibodies, then image visualization and analysis carried out by fluorescence microscopy. Treatment with 5 or 10 μM 4-AAQB markedly decreased nuclear Sox2 and Oct4 protein expression in ( A ) U87MG or ( B ) DBTRG-05MG cells. ( C ) Immunohistochemistry show reduced nuclear expression of β-catenin and F-actin after treatment with 10 μM 4-AAQB. Original magnification ×200. DAPI (blue) served as nuclear marker.

    Journal: Cancers

    Article Title: The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo

    doi: 10.3390/cancers10120491

    Figure Lengend Snippet: 4-AAQB inhibits the nuclear localization of β-catenin, Sox2, and Oct4 in GBM cells. U87MG and DBTRG-05MG cells treated with or without 5–10 μM 4-AAQB were immunostained with Sox2 (green), Oct4 (red), β-catenin (red), and F-actin (green) antibodies, then image visualization and analysis carried out by fluorescence microscopy. Treatment with 5 or 10 μM 4-AAQB markedly decreased nuclear Sox2 and Oct4 protein expression in ( A ) U87MG or ( B ) DBTRG-05MG cells. ( C ) Immunohistochemistry show reduced nuclear expression of β-catenin and F-actin after treatment with 10 μM 4-AAQB. Original magnification ×200. DAPI (blue) served as nuclear marker.

    Article Snippet: Followed by incubating the cells with antibody against β-catenin (D10A8; XP Rabbit mAb #8480; Cell Signaling Technology), Sox2 (D6D9; XP Rabbit mAb #3579; Cell Signaling Technology), Oct4 (C30A3; Rabbit mAb #2840; Cell Signaling Technology), or F-actin (13E5; Rabbit mAb #4970; Cell Signaling Technology) diluted 1:500, in PBS containing 3% normal goat serum at room temperature for 2 h. For negative controls we omitted the primary antibody.

    Techniques: Fluorescence, Microscopy, Expressing, Immunohistochemistry, Marker

    Aberrant expression of β-catenin is characteristic of glioblastoma (GBM) and correlates with poor prognosis. ( A ) Kaplan–Meier plot showing the overall survival of The Cancer Genome Atlas (TCGA) lower grade glioma and glioblastoma cohort (Glioma (GBMLGG), n = 1152) according to their histological type. ( B ) β-catenin expression across the different histological type of glioma and glioblastoma in the TCGA GBMLGG cohort. ( C ) Kaplan–Meier plot based on the β-catenin expression level in only the glioblastoma component of the TCGA GBMLGG dataset using the gene expression RNAseq-illuminaHiSeq. Expression cutoff is based on median expression. LGG, low grade glioma; GBM, glioblastoma.

    Journal: Cancers

    Article Title: The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo

    doi: 10.3390/cancers10120491

    Figure Lengend Snippet: Aberrant expression of β-catenin is characteristic of glioblastoma (GBM) and correlates with poor prognosis. ( A ) Kaplan–Meier plot showing the overall survival of The Cancer Genome Atlas (TCGA) lower grade glioma and glioblastoma cohort (Glioma (GBMLGG), n = 1152) according to their histological type. ( B ) β-catenin expression across the different histological type of glioma and glioblastoma in the TCGA GBMLGG cohort. ( C ) Kaplan–Meier plot based on the β-catenin expression level in only the glioblastoma component of the TCGA GBMLGG dataset using the gene expression RNAseq-illuminaHiSeq. Expression cutoff is based on median expression. LGG, low grade glioma; GBM, glioblastoma.

    Article Snippet: Followed by incubating the cells with antibody against β-catenin (D10A8; XP Rabbit mAb #8480; Cell Signaling Technology), Sox2 (D6D9; XP Rabbit mAb #3579; Cell Signaling Technology), Oct4 (C30A3; Rabbit mAb #2840; Cell Signaling Technology), or F-actin (13E5; Rabbit mAb #4970; Cell Signaling Technology) diluted 1:500, in PBS containing 3% normal goat serum at room temperature for 2 h. For negative controls we omitted the primary antibody.

    Techniques: Expressing

    Inhibition of Wnt/β-catenin signaling impairs ELFEF-induced increases of SVZ neurogenesis and olfactory memory. ( a – c ) Bar graphs showing the number of BrdU + ( a ) and BrdU + /Nestin + ( b ) cells in the SVZ, and BrdU + /DCX + cells ( c ) in the RMS of control and ELFEF-exposed mice perfused with either vehicle (CTRL) or Dkk-1. ( d ) In the odor discrimination test, ELFEF-exposed mice injected with vehicle (ELFEF) showed a higher discrimination ability when compared to ELFEF-exposed mice injected with Dkk-1 (Dkk-1-ELFEF). ( e ) Injection of Dkk-1 into the SVZ abolished the enhancement of olfactory memory induced by ELFEF. ( f ) In the long-term olfactory memory tests, the preference index of Dkk-1-ELFEF mice was significantly less when compared to ELFEF mice. Values are expressed as means ± SEM. (n = 7–8 mice per group). n.s. = not significant, *p

    Journal: Scientific Reports

    Article Title: Olfactory memory is enhanced in mice exposed to extremely low-frequency electromagnetic fields via Wnt/β-catenin dependent modulation of subventricular zone neurogenesis

    doi: 10.1038/s41598-017-18676-1

    Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling impairs ELFEF-induced increases of SVZ neurogenesis and olfactory memory. ( a – c ) Bar graphs showing the number of BrdU + ( a ) and BrdU + /Nestin + ( b ) cells in the SVZ, and BrdU + /DCX + cells ( c ) in the RMS of control and ELFEF-exposed mice perfused with either vehicle (CTRL) or Dkk-1. ( d ) In the odor discrimination test, ELFEF-exposed mice injected with vehicle (ELFEF) showed a higher discrimination ability when compared to ELFEF-exposed mice injected with Dkk-1 (Dkk-1-ELFEF). ( e ) Injection of Dkk-1 into the SVZ abolished the enhancement of olfactory memory induced by ELFEF. ( f ) In the long-term olfactory memory tests, the preference index of Dkk-1-ELFEF mice was significantly less when compared to ELFEF mice. Values are expressed as means ± SEM. (n = 7–8 mice per group). n.s. = not significant, *p

    Article Snippet: Rat monoclonal anti-BrdU antibody (1:400, Abcam, Cambridge, UK, Cat. #ab6326), rabbit anti-DCX antibody (1:250, Cell Signaling Cat. #4604), mouse monoclonal anti-NeuN and mouse monoclonal anti-Nestin antibodies (1:150, Millipore Cat. #MAB377 and #MAB353, respectively), rabbit polyclonal anti Ki67 and rabbit anti-β-Catenin (1:200, Abcam, Cat. #ab15580 and #ab6302, respectively) were used as primary antibodies.

    Techniques: Inhibition, Mouse Assay, Injection

    ELFEF stimulation increases β-catenin nuclear accumulation in cultured NSCs isolated from the SVZ upon differentiation. ( a – c ) Representative confocal images showing β-catenin localization (green) in control- and ELFEF-exposed NSCs at 3 h ( a ), 6 h ( b ), and 24 h ( c ) after the induction of differentiation (DM). Higher magnifications are provided in the first column (scale bar: 10 µm). Cells were counterstained with Nestin (red), and nuclei were stained with DAPI (blue) (scale bar: 25 µm). ( d . ( e ) Representative images showing β-catenin localization in NSCs from the SVZ in control and ELFEF mice. Nuclei were counterstained with DAPI (scale bar: 10 µm). Values are expressed as means ± SEM. (n = 3 per group). *p

    Journal: Scientific Reports

    Article Title: Olfactory memory is enhanced in mice exposed to extremely low-frequency electromagnetic fields via Wnt/β-catenin dependent modulation of subventricular zone neurogenesis

    doi: 10.1038/s41598-017-18676-1

    Figure Lengend Snippet: ELFEF stimulation increases β-catenin nuclear accumulation in cultured NSCs isolated from the SVZ upon differentiation. ( a – c ) Representative confocal images showing β-catenin localization (green) in control- and ELFEF-exposed NSCs at 3 h ( a ), 6 h ( b ), and 24 h ( c ) after the induction of differentiation (DM). Higher magnifications are provided in the first column (scale bar: 10 µm). Cells were counterstained with Nestin (red), and nuclei were stained with DAPI (blue) (scale bar: 25 µm). ( d . ( e ) Representative images showing β-catenin localization in NSCs from the SVZ in control and ELFEF mice. Nuclei were counterstained with DAPI (scale bar: 10 µm). Values are expressed as means ± SEM. (n = 3 per group). *p

    Article Snippet: Rat monoclonal anti-BrdU antibody (1:400, Abcam, Cambridge, UK, Cat. #ab6326), rabbit anti-DCX antibody (1:250, Cell Signaling Cat. #4604), mouse monoclonal anti-NeuN and mouse monoclonal anti-Nestin antibodies (1:150, Millipore Cat. #MAB377 and #MAB353, respectively), rabbit polyclonal anti Ki67 and rabbit anti-β-Catenin (1:200, Abcam, Cat. #ab15580 and #ab6302, respectively) were used as primary antibodies.

    Techniques: Cell Culture, Isolation, Staining, Mouse Assay

    Deletion of β-catenin suppresses growth of menin-null PNETs and increases survival time. ( a ) Immunofluorescence staining of menin, β-catenin and insulin on pancreatic sections from 8-week-old mouse models. Scale bars, 50 μm. ( b ) The incidence of tumorigenesis in mouse models ( n =18–31). * P

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: Deletion of β-catenin suppresses growth of menin-null PNETs and increases survival time. ( a ) Immunofluorescence staining of menin, β-catenin and insulin on pancreatic sections from 8-week-old mouse models. Scale bars, 50 μm. ( b ) The incidence of tumorigenesis in mouse models ( n =18–31). * P

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Immunofluorescence, Staining

    A β-catenin antagonist suppresses Men1 -deficient tumour cell proliferation and improves hypoglycemia in mice. ( a ) Body weight of βMen1-KO mice treated with PKF115-584 (0.5 mg kg −1 ) or vehicle ( n =7–8). The untreated and treated groups represent the beginning and end of treatment. ( b , c ) Fasting blood glucose and fasting serum insulin levels in βMen1-KO mice treated with PKF115-584 or vehicle ( n =6–8). The data represent the mean±s.d., * P

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: A β-catenin antagonist suppresses Men1 -deficient tumour cell proliferation and improves hypoglycemia in mice. ( a ) Body weight of βMen1-KO mice treated with PKF115-584 (0.5 mg kg −1 ) or vehicle ( n =7–8). The untreated and treated groups represent the beginning and end of treatment. ( b , c ) Fasting blood glucose and fasting serum insulin levels in βMen1-KO mice treated with PKF115-584 or vehicle ( n =6–8). The data represent the mean±s.d., * P

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Mouse Assay

    β-catenin ablation inhibits tumorigenesis of menin-null PNETs in mice. ( a ) Representative PET/CT fused images of PNETs in the βMen1-KO and βMen1/Bcat-KO mice. The arrowheads point to the tumour lesions that have high uptake of FDG. ( b ) Analysis of the average tumour volume in the βMen1-KO and βMen1/Bcat-KO mice by PET/CT ( n =3 for each group). ( c ) Analysis of the standard uptake value of 18F-FDG in mouse PNETs by PET/CT ( n =3 for each group). The data represent the mean±s.d., * P

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: β-catenin ablation inhibits tumorigenesis of menin-null PNETs in mice. ( a ) Representative PET/CT fused images of PNETs in the βMen1-KO and βMen1/Bcat-KO mice. The arrowheads point to the tumour lesions that have high uptake of FDG. ( b ) Analysis of the average tumour volume in the βMen1-KO and βMen1/Bcat-KO mice by PET/CT ( n =3 for each group). ( c ) Analysis of the standard uptake value of 18F-FDG in mouse PNETs by PET/CT ( n =3 for each group). The data represent the mean±s.d., * P

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Mouse Assay, Positron Emission Tomography

    β-catenin knockout decreases excessive insulin secretion of Men1 -deficient PNETs in mice. ( a ) Insulin levels in the pancreatic perfusion buffer in the ex vivo pancreatic glucose (16.7 mmol l −1 ) perfusion tests ( n =3–7). ( b ) Analysis of the area under curve (AUC) of the insulin levels in the ex vivo pancreatic glucose perfusion tests ( n =3–7). ( c , d ) 24-h fasting blood glucose ( c ) and serum insulin levels ( d ) in the STZ-induced diabetic mice transplanted with islets from control, βMen1-KO and βMen1/Bcat-KO mice ( n =6–8). ( e , f ) Analysis of Ki67 staining on the sections of transplanted grafts from mouse models ( n =3 for each group). The data represent the mean±s.d., * P

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: β-catenin knockout decreases excessive insulin secretion of Men1 -deficient PNETs in mice. ( a ) Insulin levels in the pancreatic perfusion buffer in the ex vivo pancreatic glucose (16.7 mmol l −1 ) perfusion tests ( n =3–7). ( b ) Analysis of the area under curve (AUC) of the insulin levels in the ex vivo pancreatic glucose perfusion tests ( n =3–7). ( c , d ) 24-h fasting blood glucose ( c ) and serum insulin levels ( d ) in the STZ-induced diabetic mice transplanted with islets from control, βMen1-KO and βMen1/Bcat-KO mice ( n =6–8). ( e , f ) Analysis of Ki67 staining on the sections of transplanted grafts from mouse models ( n =3 for each group). The data represent the mean±s.d., * P

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Knock-Out, Mouse Assay, Ex Vivo, Staining

    β-catenin knockout inhibits tumour cell proliferation and excessive insulin production in Men1- deficient mice. ( a , b ) Immunofluorescence staining and analysis of Ki67 on pancreatic sections from 6-month-old mouse models ( n =3). Scale bars, 100 μm. ( c ) Quantitative PCR analysis of Mki67 expression in the islets isolated from 6-month-old mouse models ( n =4–6). ( d ) Quantification of pancreatic β-cell mass from 4-month-old mouse models. ( e , f ) Random ( d , n =8) and fasting ( e , n =6–8) blood glucose levels in mouse models. The mice were fasted for 16 h. ( g ) Seventy-two hours fasting blood glucose levels in the 12-month-old βMen1-KO and βMen1/Bcat-KO mice ( n =13 for each group). ( h , i ) Fasting serum insulin levels in mouse models at the ages of 10 months ( h , n =6) and 15 months ( i , n =6–8). The mice were fasted for 16 h. The data represent the mean±s.d., * P

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: β-catenin knockout inhibits tumour cell proliferation and excessive insulin production in Men1- deficient mice. ( a , b ) Immunofluorescence staining and analysis of Ki67 on pancreatic sections from 6-month-old mouse models ( n =3). Scale bars, 100 μm. ( c ) Quantitative PCR analysis of Mki67 expression in the islets isolated from 6-month-old mouse models ( n =4–6). ( d ) Quantification of pancreatic β-cell mass from 4-month-old mouse models. ( e , f ) Random ( d , n =8) and fasting ( e , n =6–8) blood glucose levels in mouse models. The mice were fasted for 16 h. ( g ) Seventy-two hours fasting blood glucose levels in the 12-month-old βMen1-KO and βMen1/Bcat-KO mice ( n =13 for each group). ( h , i ) Fasting serum insulin levels in mouse models at the ages of 10 months ( h , n =6) and 15 months ( i , n =6–8). The mice were fasted for 16 h. The data represent the mean±s.d., * P

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Knock-Out, Mouse Assay, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Isolation

    Inhibition of β-catenin suppresses proproliferative gene expression in MEN1 -mutant PNETs. ( a ) Western blot analyses of Mcm2 and Pbk in human MEN1 -mutant PNETs and normal islets ( n =3). ( b ) Quantitative PCR (qPCR) analysis of MKI67 , PCNA , MCM2 , PBK , CCNA2 , CCNB2 and CCND2 in human MEN1 -mutant PNETs and normal islets ( n =3). ( c ) Western blot analyses of Mcm2, Pbk, Cyclin D2, Cyclin B2 and Cyclin A in isolated islets from mouse models. ( d ) qPCR analysis of Ccna2 , Ccnb2 , Ccnd2 , Mcm2 , Mcm6 , Myc and Pbk in isolated islets from mouse models ( n =3–6). The data represent the mean±s.d., * P

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: Inhibition of β-catenin suppresses proproliferative gene expression in MEN1 -mutant PNETs. ( a ) Western blot analyses of Mcm2 and Pbk in human MEN1 -mutant PNETs and normal islets ( n =3). ( b ) Quantitative PCR (qPCR) analysis of MKI67 , PCNA , MCM2 , PBK , CCNA2 , CCNB2 and CCND2 in human MEN1 -mutant PNETs and normal islets ( n =3). ( c ) Western blot analyses of Mcm2, Pbk, Cyclin D2, Cyclin B2 and Cyclin A in isolated islets from mouse models. ( d ) qPCR analysis of Ccna2 , Ccnb2 , Ccnd2 , Mcm2 , Mcm6 , Myc and Pbk in isolated islets from mouse models ( n =3–6). The data represent the mean±s.d., * P

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Inhibition, Expressing, Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction, Isolation

    Activation of β-catenin signalling in MEN1 -mutant human and mouse PNETs. ( a ) Western blot analyses of menin, β-catenin and active β-catenin in human MEN1 -mutant PNETs and normal islets ( n =3). ( b ) Western blot analyses of phospho-33/37/41-β-catenin (P-33/37/41) and phospho-45-β-catenin (P-45) in human MEN1 -mutant PNETs and normal islets ( n =3). ( c ) Western blot analyses of active β-catenin, phospho-33/37/41-β-catenin (P-33/37/41) and phospho-45-β-catenin (P-45) in 293T cells transfected with green fluorescent protein–β-catenin (GFP–β-catenin) and menin or vector control. The transfected cells were treated with the proteasome inhibitor MG132 (25 μM) for 2 h before the cells were harvested. The data shown represent three independent experiments. ( d ) Western blot analyses of active β-catenin and phosphorylated β-catenin in the menin-null islets from 12-month-old βMen1-KO mice and normal islets from control mice ( n =3 for each group). ( e ) Western blot analyses of cytoplasmic and nuclear β-catenin in the pancreatic islets isolated from 12-month-old βMen1-KO and control mice ( n =3 for each group).

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: Activation of β-catenin signalling in MEN1 -mutant human and mouse PNETs. ( a ) Western blot analyses of menin, β-catenin and active β-catenin in human MEN1 -mutant PNETs and normal islets ( n =3). ( b ) Western blot analyses of phospho-33/37/41-β-catenin (P-33/37/41) and phospho-45-β-catenin (P-45) in human MEN1 -mutant PNETs and normal islets ( n =3). ( c ) Western blot analyses of active β-catenin, phospho-33/37/41-β-catenin (P-33/37/41) and phospho-45-β-catenin (P-45) in 293T cells transfected with green fluorescent protein–β-catenin (GFP–β-catenin) and menin or vector control. The transfected cells were treated with the proteasome inhibitor MG132 (25 μM) for 2 h before the cells were harvested. The data shown represent three independent experiments. ( d ) Western blot analyses of active β-catenin and phosphorylated β-catenin in the menin-null islets from 12-month-old βMen1-KO mice and normal islets from control mice ( n =3 for each group). ( e ) Western blot analyses of cytoplasmic and nuclear β-catenin in the pancreatic islets isolated from 12-month-old βMen1-KO and control mice ( n =3 for each group).

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Activation Assay, Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Mouse Assay, Isolation

    β-catenin antagonist inhibits replication of menin-null tumour cell and expression of proproliferative genes in vitro . ( a , b ) Analysis of Ki67 staining on PKF115-584 (1 mM for 16 h) or vehicle-treated dispersed tumour cells from βMen1-KO mice ( n =3). Scale bars, 100 μm. ( c ) Quantitative PCR (qPCR) analysis of Mki67 expression in PKF115-584- or vehicle-treated tumour cells from βMen1-KO mice ( n =3). ( d , e ) Analysis of EdU staining on PKF115-584- or vehicle-treated dispersed tumour cells from βMen1-KO mice ( n =3). Scale bars, 100 μm. ( f , g ) Western blot analyses of cytoplasmic and nuclear β-catenin in PKF115-584- or vehicle-treated tumour cells from βMen1-KO mice ( n =3). ( h , i ) qPCR and Western blot analyses of Mcm2 , Pbk , Ccna2 , Ccnb2 , Ccnd2 , Mcm6 and Myc expression in PKF115-584- or vehicle-treated tumour cells from βMen1-KO mice ( n =3). ( j ) ChIP assays were performed to show the binding of β-catenin at the promoters of Ccnd1 , Myc and Mcm2 in vehicle or PKF115-584 (1 mM for 16 h) treated tumour cells from βMen1-KO mice ( n =3). The pancreatic islets were isolated from 12-month-old βMen1-KO and βMen1/Bcat-KO mice. Sonicated cell lysates were incubated with β-catenin antibody for protein–DNA binding detection. Triplicate qPCR reactions for each sample showed the results from 660–400 bp (p2) and 2,750–2,552 bp (p4) of upstream regions of the Ccnd1 promoter; 1,030–885 bp (p3) of the Myc promoter; 712–585 bp (p3) and 1,428–1,326 bp (p5) of the Mcm2 promoter. All data are normalized against immunoglobulin-G control and expressed as percentage of input. The data represent the mean±s.d., * P

    Journal: Nature Communications

    Article Title: Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours

    doi: 10.1038/ncomms6809

    Figure Lengend Snippet: β-catenin antagonist inhibits replication of menin-null tumour cell and expression of proproliferative genes in vitro . ( a , b ) Analysis of Ki67 staining on PKF115-584 (1 mM for 16 h) or vehicle-treated dispersed tumour cells from βMen1-KO mice ( n =3). Scale bars, 100 μm. ( c ) Quantitative PCR (qPCR) analysis of Mki67 expression in PKF115-584- or vehicle-treated tumour cells from βMen1-KO mice ( n =3). ( d , e ) Analysis of EdU staining on PKF115-584- or vehicle-treated dispersed tumour cells from βMen1-KO mice ( n =3). Scale bars, 100 μm. ( f , g ) Western blot analyses of cytoplasmic and nuclear β-catenin in PKF115-584- or vehicle-treated tumour cells from βMen1-KO mice ( n =3). ( h , i ) qPCR and Western blot analyses of Mcm2 , Pbk , Ccna2 , Ccnb2 , Ccnd2 , Mcm6 and Myc expression in PKF115-584- or vehicle-treated tumour cells from βMen1-KO mice ( n =3). ( j ) ChIP assays were performed to show the binding of β-catenin at the promoters of Ccnd1 , Myc and Mcm2 in vehicle or PKF115-584 (1 mM for 16 h) treated tumour cells from βMen1-KO mice ( n =3). The pancreatic islets were isolated from 12-month-old βMen1-KO and βMen1/Bcat-KO mice. Sonicated cell lysates were incubated with β-catenin antibody for protein–DNA binding detection. Triplicate qPCR reactions for each sample showed the results from 660–400 bp (p2) and 2,750–2,552 bp (p4) of upstream regions of the Ccnd1 promoter; 1,030–885 bp (p3) of the Myc promoter; 712–585 bp (p3) and 1,428–1,326 bp (p5) of the Mcm2 promoter. All data are normalized against immunoglobulin-G control and expressed as percentage of input. The data represent the mean±s.d., * P

    Article Snippet: The protein levels of active β-catenin in whole-cell lysates were quantified using the anti-Active β-catenin clone 8E7 antibody (Millipore).

    Techniques: Expressing, In Vitro, Staining, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Isolation, Sonication, Incubation