Journal: Respiratory Research

Article Title: ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

doi: 10.1186/s12931-020-01568-w

Figure Lengend Snippet: Effect of ROBO1 or ROBO2 functional impairment in branching morphogenesis. a The upper panel is representative of untreated lung explants (0 ng/mL) at D0; the bottom panel represents lung explants treated with recombinant IgG protein (0 ng/mL) and several doses of recombinant ROBO1 or ROBO2 proteins at day 4 (D4). b Morphometric analysis of the number of peripheral airway buds of fetal rat lung explants treated with increasing concentrations of recombinant ROBO1 (black) and ROBO2 (gray) proteins. Results are expressed as the D4/D0 ratio. c Examples of representative blots are showed for each analysed protein. Protein expression levels of d ROBO; e SOX2; f SOX9; g total β-Catenin; h non-phospho (active) β-Catenin; and i BMP4 in normal explant cultures treated with recombinant rat ROBO1 Fc Chimera for ROBO1 inhibition (black line) or recombinant human ROBO2 Fc Chimera for ROBO2 functional impairment (gray line). Recombinant human IgG Fc was used as control (IgG). n ≥ 9 per protein/condition. Each lane represents a pooled-tissue sample, and relative expression was determined against β-Tubulin and IgG. The data are presented as means ± SEM. p < 0.05: α vs. IgG

Article Snippet: Blots were blocked in 5% bovine serum albumin and probed with primary antibodies to ROBO1 (1:500, ON, 4 oC; Cat No. sc25672, Santa Cruz Biotechnology Inc., USA), ROBO2 (1:250, ON, 4 oC; Cat No. sc16615, Santa Cruz Biotechnology Inc. USA), SOX2 (1:250, ON, 4 oC; Cat No. AF2018, R&D system, USA), SOX9 (1:250, ON, 4 oC; Cat No. AF3075, R&D system, USA), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (1∶5000; Cat No. #4270, Cell Signaling Technology Inc., USA), total β-Catenin (1∶30,000; Cat No. #NBP1-54,467, NOVUS Biologicals, USA), and BMP4 (1:250, ON, 4 oC; Cat No. sc-6896, Santa Cruz Biotechnology Inc., USA) according to the manufacturer's instructions.

Techniques: Functional Assay, Recombinant, Expressing, Inhibition, Control

Journal: Respiratory Research

Article Title: ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

doi: 10.1186/s12931-020-01568-w

Figure Lengend Snippet: a Overview of the main changes in spatiotemporal distribution of ROBO1, ROBO2, SOX2 and SOX9 at pseudoglandular (E17.5) and saccular stages (E21.5) in hypoplastic (hyp) fetal lungs (lower panel). w/o without b A proposed model of ROBO regulation of ex vivo branching morphogenesis through BMP4, β-Catenin, SOX2 and SOX9

Article Snippet: Blots were blocked in 5% bovine serum albumin and probed with primary antibodies to ROBO1 (1:500, ON, 4 oC; Cat No. sc25672, Santa Cruz Biotechnology Inc., USA), ROBO2 (1:250, ON, 4 oC; Cat No. sc16615, Santa Cruz Biotechnology Inc. USA), SOX2 (1:250, ON, 4 oC; Cat No. AF2018, R&D system, USA), SOX9 (1:250, ON, 4 oC; Cat No. AF3075, R&D system, USA), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (1∶5000; Cat No. #4270, Cell Signaling Technology Inc., USA), total β-Catenin (1∶30,000; Cat No. #NBP1-54,467, NOVUS Biologicals, USA), and BMP4 (1:250, ON, 4 oC; Cat No. sc-6896, Santa Cruz Biotechnology Inc., USA) according to the manufacturer's instructions.

Techniques: Ex Vivo

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Macrophage β-catenin is activated in HFD-induced MASH. Wild-type mice were provided with either a normal chow diet (NCD) or a high-fat diet (HFD) for 28 weeks. A Double immunofluorescence staining was performed on liver sections. Antibodies against F4/80 (displayed in green) and A-β-catenin (shown in red) were used. The cell nuclei were stained with DAPI (appearing blue). Five mice were included in each group. The scale bar represents 200 μm. B Liver macrophages were separated from mice that had been fed NCD or HFD. The mRNA level of Ctnnb1 (the gene encoding β-catenin) in liver macrophages of wild-type mice that had received NCD or HFD for 28 weeks was measured ( n = 5 samples/group). C Western blot analysis was carried out to detect the expression of A-β-catenin in liver macrophages from wild-type mice fed with NCD or HFD ( n = 5 samples/group). The data are presented as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Double Immunofluorescence Staining, Staining, Western Blot, Expressing, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Deficiency of myeloid β-catenin alleviates hepatic steatosis induced by HFD. A Serum ALT and AST levels were measured in β-catenin FL/FL and β-catenin M−KO mice following 28 weeks of normal chow diet (NCD) or high-fat diet (HFD) feeding ( n = 5 samples/group). B Liver-to-body weight ratios were examined in β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). C Liver triglyceride (TG) and total cholesterol (TC) levels were determined in β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). D Representative hematoxylin and eosin (H&E) staining of liver sections and the non-alcoholic fatty liver disease activity score (NAS) were obtained from β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). The scale bars represent 100 μm. E Representative Oil Red O-stained images of liver sections were taken from β-catenin FL/FL and β-catenin M−KO mice fed with NCD or HFD ( n = 5 samples/group). The scale bar is 50 μm. The data are presented as mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Staining, Activity Assay, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Deficiency of myeloid β-catenin ameliorates MASH-associated liver fibrosis. A Representative Sirius Red staining of liver sections was shown from β-catenin FL/FL and β-catenin M−KO mice. These mice were fed with either a normal chow diet (NCD) or a high-fat diet (HFD) for 28 weeks ( n = 5 samples/group). Scale bars were 100 μm. B Immunofluorescence images of α-SMA + myofibroblasts in liver sections were obtained from NCD-fed or HFD-fed β-catenin FL/FL and β-catenin M−KO mice ( n = 5 samples/group). Scale bars measured 50 μm. C Relative mRNA levels of fibrogenic genes such as Acta2 , Col1α1 , Col3α 1, and Timp 1 were detected in the livers of β-catenin FL/FL and β-catenin M−KO mice that were fed with NCD or HFD ( n = 5 samples/group). Data were expressed as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Staining, Immunofluorescence, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Deficiency of myeloid β-catenin inhibits Ihh expression in macrophages in HFD-induced MASH. A Relative mRNA levels of Shh and Ihh in liver macrophages isolated from HFD-fed β-catenin FL/FL and β-catenin M−KO mice were determined. B Western blot was performed to analyze the expression of Ihh in liver macrophages from HFD-fed β-catenin FL/FL and β-catenin M−KO mice. C Double immunofluorescence staining was carried out on liver sections using antibodies against F4/80 (green) and Ihh (red). The nuclei were stained with DAPI (blue). There were five mice in each group. The scale bar represents 200 μm. The data are shown as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Expressing, Isolation, Western Blot, Double Immunofluorescence Staining, Staining, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Ihh signaling is crucial for macrophage β-catenin-mediated hepatic fibrosis in HFD-induced MASH. To restore Ihh expression in macrophages, mice were administered the Ihh plasmid via polyethylenimine nanoparticles, namely in vivo jetPEI-Man. A Representative Sirius Red staining images of liver sections from HFD-fed β-catenin FL/FL and β-catenin M−KO mice. These mice were either treated or not treated with jetPEI-Man-Ihh (jetPEI-Ihh) or jetPEI-Man-GFP (jetPEI-GFP) ( n = 5 mice per group). Scale bars are 100 μm. B Immunofluorescence images of α-SMA + myofibroblasts in liver sections from HFD-fed β-catenin FL/FL and β-catenin M−KO mice. These mice were either treated or not treated with jetPEI-Ihh or jetPEI-GFP ( n = 5 mice per group). Scale bars measure 50 μm. C Relative mRNA levels of fibrogenic genes such as Acta2 , Col1α1 , Col3α1 , and Timp1 in the livers of HFD-fed β-catenin FL/FL and β-catenin M−KO mice. These mice were either treated or not treated with jetPEI-Ihh or jetPEI-GFP ( n = 5 mice per group). Data are expressed as the mean ± standard deviation (SD). * P < 0.05, ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Expressing, Plasmid Preparation, In Vivo, Staining, Immunofluorescence, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Macrophage β-catenin induces Ihh expression and secretion. Bone marrow-derived macrophages (BMDMs) were treated with LPS (100 ng/ml) and PA (250 µM) or PBS for 12 h. A The mRNA level of Ctnnb1 in BMDMs following treatment with PA/LPS or PBS. B Western blot analysis was carried out to assess the expression of A-β-catenin in BMDMs after treatment with PA/LPS or PBS. C BMDMs underwent a ChIP-PCR assay using an anti-β-catenin or IgG antibody to detect the potential β-catenin binding site within the Ihh intron 1. D The mRNA level of Ihh in BMDMs isolated from β-catenin FL/FL and β-catenin M−KO mice. E The concentration of Ihh in the media of BMDMs isolated from β-catenin FL/FL and β-catenin M−KO mice was measured by ELISA. The data are presented as the mean ± standard deviation (SD). ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Expressing, Derivative Assay, Western Blot, Binding Assay, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Macrophage β-catenin-induced Ihh promotes HSC activation. A The schematic illustration of the co-culture model that utilizes bone marrow-derived macrophages (BMDMs) and primary murine hepatic stellate cells (HSCs). B The relative mRNA levels of fibrogenic genes such as Acta2 , Col1α1 , and Col3α1 in HSCs were measured after co-culturing with BMDMs from β-catenin FL/FL and β-catenin M−KO mice. C Immunofluorescence pictures of α-SMA expression in HSCs were obtained after co-culturing with BMDMs from β-catenin FL/FL and β-catenin M−KO mice. The scale bars represent 100 μm. D Immunofluorescence images of α-SMA expression in HSCs were captured after co-culturing with BMDMs transfected with the Lv-β-catenin plasmid or the control vector (Lv-GFP). The scale bars are 100 μm. E Immunofluorescence images of α-SMA expression in HSCs were taken after co-culturing with Lv-β-catenin-transfected BMDMs, either in the presence or absence of Ihh neutralizing antibody. The scale bars measure 100 μm. F The mRNA levels of Gli1 , Gli2 , and Gli3 in HSCs were determined after co-culturing with BMDMs from β-catenin FL/FL and β-catenin M−KO mice. The data are presented as the mean ± standard deviation (SD). * P < 0.05, ** P < 0.01

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Activation Assay, Co-Culture Assay, Derivative Assay, Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Inflammation Research

Article Title: Macrophage β-catenin-Ihh axis induces hepatic stellate cell activation and fibrosis in metabolic dysfunction-associated steatohepatitis

doi: 10.1007/s00011-026-02220-x

Figure Lengend Snippet: Schematic representation of the macrophage β-catenin-Ihh axis in modulating the activation of hepatic stellate cells and fibrosis in MASH. The consumption of a high-fat diet (HFD) led to the activation of β-catenin in liver macrophages. The activation of macrophage β-catenin triggered the expression and secretion of Ihh, which in turn facilitated the activation of hepatic stellate cells and fibrosis in MASH

Article Snippet: By using rat monoclonal antibody for F4/80 (Cat#: sc-52664, dilution 1:50, Santa Cruz Biotechnology, California) as well as rabbit monoclonal antibody for active β-catenin (non-phosphorylated serine 33/37/threonine41) (Cat#: 8814, 1:200 dilution, Cell Signaling Technology, Massachusetts) or anti-Ihh antibody (Cat#: ab39634, 1:100 dilution, Abcam, Massachusetts), the double-positive macrophages for F4/80 and A-β-catenin or Ihh were successfully identified.

Techniques: Activation Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling

doi: 10.3390/ijms23052834

Figure Lengend Snippet: β-catenin pathway mediates neurite outgrowth in PC12 cells. ( A ) Effect of β-catenin inhibitors on TNKS1/2 localization and neurite outgrowth in PC12 cells. Cells were treated with ICG-001 (5 μM) and PNU-74654 (20 μM) after 1 day in culture with NGF (100 ng/mL). After 4 days, cells were stained with anti-TNKS1/2 and anti-MAP2 antibodies. Nuclei were stained with DAPI. Scale bar: 20 μm. ( B ) Mean neurite length in PC12 cells. Data are shown as means ± SEM ( n = 14–18). *** p < 0.001 vs. Control (one-way ANOVA with post hoc Dunnett’s test).

Article Snippet: PJ34 was purchased from Enzo Life Sciences (Farmingdale, NY, USA); ABT888 from APExBIO (Houston, TX, USA); XAV939 from Cayman chemical company (Ann Arbor, MI, USA); MG-132, ICG-001, and PNU-74654 from Selleckchem (Houston, TX, USA); nerve growth factor-7S (NGF) from Sigma-Aldrich (Burlington, MA, USA); rabbit polyclonal anti-TNKS1/2 antibody (H-350), mouse monoclonal anti-MAP2 antibody (AP20), mouse monoclonal anti-PAR antibody (10H), and ADP-HPD from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit monoclonal anti-Axin1 antibody (C76H11), and rabbit monoclonal anti-unphosphorylated (Ser33/Thr41) β-catenin antibody (D13A1) from Cell Signaling Technology (Danvers, MA); DAPI, Alexa Fluor 488-conjugated goat anti-rabbit IgG, and Alexa Fluor 568-conjugated goat anti-mouse IgG from Thermo Fisher Scientific (Waltham, MA, USA)); and predesigned primers for real-time PCR from Takara Bio (Kusatsu, Japan).

Techniques: Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling

doi: 10.3390/ijms23052834

Figure Lengend Snippet: TNKS1/2 regulate β-catenin signaling through poly(ADP-ribosyl)ation in PC12 cells. ( A ) Effect of XAV939 on protein expression of Axin1 and unphosphorylated (non- p )-β-catenin in PC12 cells. Cells were treated with XAV939 (10 μM) for 1 day, followed by Western blotting with the indicated antibodies. GAPDH was used as a loading control. ( B ) Relative protein expression of Axin1 and non-p-β-catenin. Data are shown as means ± SEM ( n = 3). ** p < 0.01 vs. Control (Student’s t -test). ( C ) Effect of XAV939 on subcellular localization of non-p-β-catenin in PC12 cells. Cells were treated for 1 day with XAV939 (10 μM) and then stained using an anti-non-p-β-catenin antibody. Nuclei were stained with DAPI. Scale bar: 20 μm. ( D ) and ( F ) Effect of XAV939 on poly(ADP-ribosyl)ation of TNKS1/2 ( D ) and Axin1 ( F ) in PC12 cells. Cells were treated for 4 h with XAV939 (10 μM) and then subjected to macrodomain (MacroD)-GST pulldown assays followed by Western blotting with the indicated antibodies. ( E ) and ( G ) Relative poly(ADP-ribosyl)ation levels on TNKS1/2 ( E ) and Axin1 ( G ). Data are shown as means ± SEM ( n = 4). * p < 0.05, *** p < 0.001 vs. Control (Student’s t -test). ( H ) Effect of PARP inhibitors on NrCAM mRNA expression in PC12 cells. Cells were treated for 24 h with the indicated PARP inhibitors (10 μM) and then subjected to real-time PCR using NrCAM primers. NrCAM mRNA levels were normalized to GAPDH mRNA. Data are shown as means ± SEM ( n = 3) * p < 0.05, *** p < 0.001 vs. Control (one-way ANOVA with post hoc Dunnett’s test).

Article Snippet: PJ34 was purchased from Enzo Life Sciences (Farmingdale, NY, USA); ABT888 from APExBIO (Houston, TX, USA); XAV939 from Cayman chemical company (Ann Arbor, MI, USA); MG-132, ICG-001, and PNU-74654 from Selleckchem (Houston, TX, USA); nerve growth factor-7S (NGF) from Sigma-Aldrich (Burlington, MA, USA); rabbit polyclonal anti-TNKS1/2 antibody (H-350), mouse monoclonal anti-MAP2 antibody (AP20), mouse monoclonal anti-PAR antibody (10H), and ADP-HPD from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit monoclonal anti-Axin1 antibody (C76H11), and rabbit monoclonal anti-unphosphorylated (Ser33/Thr41) β-catenin antibody (D13A1) from Cell Signaling Technology (Danvers, MA); DAPI, Alexa Fluor 488-conjugated goat anti-rabbit IgG, and Alexa Fluor 568-conjugated goat anti-mouse IgG from Thermo Fisher Scientific (Waltham, MA, USA)); and predesigned primers for real-time PCR from Takara Bio (Kusatsu, Japan).

Techniques: Expressing, Western Blot, Control, Staining, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling

doi: 10.3390/ijms23052834

Figure Lengend Snippet: Tankyrase activates β-catenin signaling to enhance neurite outgrowth and synapse formation. TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin and TNKS1/2 themselves, which activates β catenin signaling. Once translocated to the nucleus, β-catenin promotes transcription of NrCAM gene. β-catenin is also involved in stabilizing cadherin on the cell membrane. TNKS1/2-mediated upregulation of adhesion molecules may enhance neurite outgrowth and synapse formation.

Article Snippet: PJ34 was purchased from Enzo Life Sciences (Farmingdale, NY, USA); ABT888 from APExBIO (Houston, TX, USA); XAV939 from Cayman chemical company (Ann Arbor, MI, USA); MG-132, ICG-001, and PNU-74654 from Selleckchem (Houston, TX, USA); nerve growth factor-7S (NGF) from Sigma-Aldrich (Burlington, MA, USA); rabbit polyclonal anti-TNKS1/2 antibody (H-350), mouse monoclonal anti-MAP2 antibody (AP20), mouse monoclonal anti-PAR antibody (10H), and ADP-HPD from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit monoclonal anti-Axin1 antibody (C76H11), and rabbit monoclonal anti-unphosphorylated (Ser33/Thr41) β-catenin antibody (D13A1) from Cell Signaling Technology (Danvers, MA); DAPI, Alexa Fluor 488-conjugated goat anti-rabbit IgG, and Alexa Fluor 568-conjugated goat anti-mouse IgG from Thermo Fisher Scientific (Waltham, MA, USA)); and predesigned primers for real-time PCR from Takara Bio (Kusatsu, Japan).

Techniques: Membrane

Journal: eLife

Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

doi: 10.7554/eLife.38314

Figure Lengend Snippet: The biological properties of the newly defined HAVCR2 high LSC subgroup in comparison to other subgroups in the Pten -null T-ALL model.

Article Snippet: Antibody , Nonphospho (active)-β- catenin , Cell Signaling Technology , 70034 s , .

Techniques: Comparison, Marker, Activity Assay

Journal: eLife

Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

doi: 10.7554/eLife.38314

Figure Lengend Snippet: ( A ) q-PCR analysis of SPI1 and SPI1-regulated HAVCR2 and c-MYC expressions after the overexpression of active β-catenin in the Jurkat T-ALL cell line (red bars). The data are normalized to that of empty plasmid controls (blue bars); ( B–E ) Upper panels: quantitative intracellular FACS analyses of P-GSK-3β, non-phospho-β-catenin, P-p65 and SPI1 levels in the HAVCR2 high , HAVCR2 low and blast subgroups; lower panels: representative intracellular FACS analysis of P-GSK-3β, non-phospho-β-catenin, P-p65 and SPI1 levels in the HAVCR2 high , HAVCR2 low and blast subgroups. Gray line, isotype control;( F ) FACS analysis shows cells in the HAVCR2 high subgroup at the ETP/DN1 stage, which are absent in WT and dKO mice; ( G ) Representative FACS plots show the number of cells in the HAVCR2 high subgroup in the different drug treatment groups. The data in A, B, C, D and E are the means ± S.Ds of 3 independent tests; *p≤0.05; **p≤0.01; ***p≤0.001.

Article Snippet: Antibody , Nonphospho (active)-β- catenin , Cell Signaling Technology , 70034 s , .

Techniques: Over Expression, Plasmid Preparation, Control

Journal: eLife

Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

doi: 10.7554/eLife.38314

Figure Lengend Snippet: ( A ) Comparison of the HAVCR2 high subgroup population (left panel) and of the levels of non-phosphorylated β -catenin (middle panel) and SPI1 (right panel) within the HAVCR2 high subgroup without (upper panels) and with BAY6060 treatment (low panels); ( B ) Survival curve for Cdh5-Cre + ;Pten L/L mice treated with BAY6060 and BAY1082439 alone and in combination; ( C ) Survival curve for Cdh5-Cre + ;Pten L/L mice treated with rapamycin in combination with either an IgG control antibody or an anti-HAVCR2 antibody.

Article Snippet: Antibody , Nonphospho (active)-β- catenin , Cell Signaling Technology , 70034 s , .

Techniques: Comparison, Control

Journal: eLife

Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

doi: 10.7554/eLife.38314

Figure Lengend Snippet:

Article Snippet: Antibody , Nonphospho (active)-β- catenin , Cell Signaling Technology , 70034 s , .

Techniques:

Journal: Cell Death & Disease

Article Title: TGF- β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion

doi: 10.1038/cddis.2014.395

Figure Lengend Snippet: ZEB1 mediates TGF- β -induced mesenchymal transdifferentiation in GBM cells. ( a ) Western blots showing the effect of TGF- β administration on the expression of the indicated proteins, illustrating enhanced expression of the transcription factor ZEB1 in association with the upregulation of the mesenchymal markers Fibronectin, COL5A1 and MMP9 in U87 and U251 cells. The canonical Wnt signaling conferring transcription factor β -Catenin is also induced by TGF- β in U87 cells. Representative blots are shown of n =3. ( b ) A8301 prevents TGF- β -induced ZEB1 expression in GBM cells and inhibits β -Catenin accumulation in U87 cells. Consistently, induction of mesenchymal marker expression is prevented. Representative blots are shown of n =3. ( c ) Immunofluorescence microscopy ( × 40) showing accumulation and nuclear localization of ZEB1 in U87 cells. β -Catenin remains in the cytoplasm. ( d ) Phase contrast microscopy ( × 10) showing that TGF- β -induced acquisition of a mesenchymal morphology in U87 cells is prevented by siRNA-mediated silencing of ZEB1. Three different siRNAs directed against ZEB1 were used, of which the indicated two (siZEB1-I and siZEB1-II) were effective in blocking TGF- β -induced mesenchymal transdifferentiation when compared with siControl-transfected and mock-treated cells. ( e ) Expression levels were quantified with qRT-PCR and the bars represent the mean of in general three independent experiments measured in triplicate±S.E.M. (* P <0.05 and *** P <0.001, siZEB1-I and siZEB1-II versus scramble siRNA). ( f ) Wound healing assays showing reduced TGF- β -induced migratory activity upon silencing of ZEB1 using siZEB1-I or siZEB1-II. Quantified results of three independent results are shown in ( g ) (** P <0.01 and *** P <0.001, siZEB1-I and siZEB1-II versus scramble siRNA)

Article Snippet: Subsequently, cells were incubated with the indicated primary antibodies at room temperature for 1.5 h. Primary antibodies used were as follows: purified mouse anti-Fibronectin (1 : 50; 610077; BD Transduction Laboratories, San Jose, CA, USA), anti-COL5A1 (1 : 200; sc-20648; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PDGFR- α (1 : 500; ab61219; Abcam, Cambridge, UK), anti-EGFR (Merck Millipore), anti-ZEB1 (1 : 50; sc-10572; Santa Cruz Biotechnology Inc.), Anti-Active- β -Catenin (1 : 100; 05-665; Merck Millipore).

Techniques: Western Blot, Expressing, Marker, Immunofluorescence, Microscopy, Blocking Assay, Transfection, Quantitative RT-PCR, Activity Assay

Journal: Cell Death & Disease

Article Title: β-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression

doi: 10.1038/s41419-020-2687-6

Figure Lengend Snippet: a – c HUVEC were transfected with siRNA targeting β-catenin (100 nM) or scrambled control siRNA (Scr) and cultured for 72 h before treatment for 5 min with vehicle or histamine (100 mmol/L). Cell lysates were analysed by western blot using total eNOS, b-catenin, phospho-Ser1177 ( a ) or phospho-Ser633 ( b ) antibodies. c cGMP levels were quantified by ELISA and results expressed relative to protein content per sample (shown relative to scrambled control; n = 4). d – f HUVEC were cultured for 72 h before treatment for the indicated times with vehicle or LiCl (20 mM). Cell lysates were analysed by western blot using total eNOS, phospho-Ser1177 ( d ) or phospho-Ser633 ( e ) and b-catenin ( f ) antibodies. a , b , d , e Results expressed as the densitometric ratio of phospho-eNOS/GAPDH to total eNOS/GAPDH and shown relative to untreated control ( n = 5); analysis by one-way ANOVA with repeated measures, ns non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Cells were incubated overnight at 4 °UF C with anti-cleaved caspase-3 antibody (cell signalling), active β-catenin (BD biosciences) or VE-Cadherin (BD Biosciences) or active β-catenin (Millipore) and/or 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei.

Techniques: Transfection, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: β-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression

doi: 10.1038/s41419-020-2687-6

Figure Lengend Snippet: a Wild-type and β-catenin −/− MPECs were treated with SNAP (10 µM) or vehicle. b HUVEC were transfected with siRNA targeting β-catenin (100 nM) or scrambled control and cultured for 72 h before treatment with TNFα (10 ng/ml) for 18 h in the presence or absence of SNAP (10 µM). a , b EC were fixed and incubated with anti-cleaved caspase-3 antibody and DAPI. Representative images are shown. The percentage of cleaved caspase-3 positive cells was calculated in five randomly selected fields of view ( n = 4). c HUVEC were transfected with siRNA targeting β-catenin (100 nM) or scrambled control and cultured for 72 h before treatment with H 2 O 2 (200 µM) for 6 h in the presence or absence of sildenafil (50 nM; n = 4). The bands shown by way of example are from the same experiment. d HUVEC were treated with H 2 O 2 (200 µM) for 6 h in the presence of SNAP (10 µM), ODQ (10 µM) or both in combination ( n = 3). c , d Cleaved (17 and 19 kDa) and full length (35 kDa) caspase-3 were detected in western blots of cell lysates and normalised to β-actin. Densitometry values are shown expressed relative to vehicle control. e HUVEC were transfected with siRNA targeting β-catenin (100 nM) or scrambled control and cultured for 72 h before treatment with H 2 O 2 (200 µM) for 6 h in the presence or absence of SNAP (10 µM, following which cells were fixed and DNA fragmentation assessed using a Click-IT TUNEL Imaging Kit ( n = 5). All analyses by one-way ANOVA with repeated measures, ns non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Scale bars show 100 mm.

Article Snippet: Cells were incubated overnight at 4 °UF C with anti-cleaved caspase-3 antibody (cell signalling), active β-catenin (BD biosciences) or VE-Cadherin (BD Biosciences) or active β-catenin (Millipore) and/or 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei.

Techniques: Transfection, Cell Culture, Incubation, Western Blot, TUNEL Assay, Imaging

Journal: Cell Death & Disease

Article Title: β-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression

doi: 10.1038/s41419-020-2687-6

Figure Lengend Snippet: a HUVEC were exposed to orbital flow for 72 h, fixed and subject to PLA using antibodies targeting eNOS and b-catenin. In control samples, PLA was carried out in the absence of b-catenin (I) or eNOS antibody (II). VE-cadherin and DAPI were used to stain cell junctions and nuclei, respectively. Representative images are shown and the average PLA signal quantified in HUVEC under disturbed flow (DF) or undisturbed flow (UF; n = 4); analysis by paired Student’s t test, ** p ≤ 0.01. b EC from protected (outer curvature) or atherosusceptible (inner curvature) regions of the mouse aorta were stained with eNOS (red) and β-catenin (green) antibodies and imaged en face by confocal microscopy. Merged representative images with DAPI (blue) are shown ( n = 3). Scale bars show 50 mm.

Article Snippet: Cells were incubated overnight at 4 °UF C with anti-cleaved caspase-3 antibody (cell signalling), active β-catenin (BD biosciences) or VE-Cadherin (BD Biosciences) or active β-catenin (Millipore) and/or 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei.

Techniques: Staining, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: β-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression

doi: 10.1038/s41419-020-2687-6

Figure Lengend Snippet: a Cell lysates were obtained from HUVEC exposed to UF or DF for 72 h ( n = 4). b EC transfected with non-targeting scrambled or β-catenin targeting siRNA (100 nM) were exposed to orbital flow for 72 h ( n = 5). c Cell lysates obtained from HUVEC exposed to UF or DF for 72 h and treated with vehicle or LiCl (20 mM) for the last 4 h ( n = 3). a – c Cell lysates were analysed by western blot with the indicated antibodies. Results are expressed as the densitometric ratio of phospho-eNOS/eNOS to total eNOS/calnexin and shown relative to levels in UF in vehicle or scr treated conditions; analysis by paired Student’s t test ( a ) or analysis by one-way ANOVA with repeated measures ( b , c ), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Cells were incubated overnight at 4 °UF C with anti-cleaved caspase-3 antibody (cell signalling), active β-catenin (BD biosciences) or VE-Cadherin (BD Biosciences) or active β-catenin (Millipore) and/or 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei.

Techniques: Transfection, Western Blot

Journal: Cell Death & Disease

Article Title: β-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression

doi: 10.1038/s41419-020-2687-6

Figure Lengend Snippet: a – e HUVEC were exposed to orbital flow for 72 h and treated with DMSO, ODQ (10 mM) ( a ), iCRT5 (50 mM) ( d ) or Histamine (100 mmol/L) ( e ) for the last 24 h of flow exposure or transfected with b-catenin or Scr siRNA (100 nM) ( b ). b ECs were fixed and incubated with a cleaved caspase-3 antibody (green), anti-b-catenin (red) and nuclei stained with DAPI. Representative images are shown. a , b , d , e The percentage of cleaved caspase-3 positive cells was quantified in regions of undisturbed (UF) or disturbed flow (DF) ( n = 3–7). c HUVEC were exposed to orbital flow or static conditions for 72 h. Full length (35 kDa) caspase-3 was detected in western blots of cell lysates. Results are expressed as the densitometric ratio of caspase-3 to calnexin. Representative western blots are shown (right panel) ( n = 9); analysis by paired Student’s t test c or analysis by one-way ANOVA with repeated measures ( a , b , d , e ), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Scale bars show 50 mm.

Article Snippet: Cells were incubated overnight at 4 °UF C with anti-cleaved caspase-3 antibody (cell signalling), active β-catenin (BD biosciences) or VE-Cadherin (BD Biosciences) or active β-catenin (Millipore) and/or 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei.

Techniques: Transfection, Incubation, Staining, Western Blot

Journal: JBMR Plus

Article Title: The anti-HIV drug abacavir stimulates β-catenin activity in osteoblast lineage cells

doi: 10.1093/jbmrpl/ziae037

Figure Lengend Snippet: Total and active β-catenin protein expression and the mRNA expression of Wnt/β-catenin related genes in HOBs treated with ABC. (a) Representative total and hypo-phosphorylated (active) β-catenin western blot with 0.25 and 0.5 μg/ml of ABC or DMSO. (b) Representative total and active β-catenin western blot with 0.5 and 4.0 μg/ml of ABC or DMSO. (c) mRNA expression of Tcf3, Tcf4, Lef1, Axin2, Dkk1, Tnfrsf11b (osteoprotegrin, Opg), Sost . Relative mRNA expression is presented as mean ± standard deviation of 3 independent experiments (biological replicates) each performed in triplicate (technical replicates). Data were analyzed with a one-way analysis of variance (ANOVA) followed by post-hoc comparisons, when appropriate. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Following fixation, cells were stained to assess total β-catenin (1:100, Sigma-Aldrich), hypo-phosphorylated (active) β-catenin (1:100, USBiological), anti-rabbit α-tubulin (1:100, Cell Signaling), or anti-mouse α-tubulin (1:100, Boster Bio).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Aging Cell

Article Title: HIV and drug abuse mediate astrocyte senescence in a β‐catenin‐dependent manner leading to neuronal toxicity

doi: 10.1111/acel.12593

Figure Lengend Snippet: Meth and HIV infection decrease β‐catenin signaling. A) HFA s were transfected with TOP flash and treated daily with 300 μ m of meth. At 48 h post‐transfection, luciferase activities were measured. The value of arbitrary units per μg of cellular protein in the control was set at 100%. B‐C) HFA s were infected with VSVG ‐ HIV . Cell lysates were collected postinfection at 0, 24, 48, and 72 h. Western analysis was used for the detection of total β‐catenin and active β‐catenin, normalized to GAPDH . * denotes P < 0.05. n = 3–5.

Article Snippet: Other antibodies include Iba1 antibody (Fisher Scientific #019‐19741, Pittsburgh, PA), active β‐catenin antibody (US Biological C2069‐47, Salem, MA), total β‐catenin antibody (C2206; Sigma), p16 INK4A antibody (Proteintech #22515‐1‐AP, Chicago, IL), GAPDH antibody (G99445; Sigma), antineurofilament heavy chain (Millipore Ab5539, Danvers, MA).

Techniques: Infection, Transfection, Luciferase, Control, Western Blot

Journal: Aging Cell

Article Title: HIV and drug abuse mediate astrocyte senescence in a β‐catenin‐dependent manner leading to neuronal toxicity

doi: 10.1111/acel.12593

Figure Lengend Snippet: Suppression of β‐catenin induces HFA senescence (A, B). A) HFA s were transfected with scrambled control si RNA or β‐catenin si RNA . At day 3, cell lysates were harvested for analysis of β‐catenin levels by Western blotting, using GAPDH as control. B) In parallel experiments, si RNA ‐transfected HFA s were treated with 1x PBS vehicle or 300 μ m of meth daily. At day 6, cells were fixed and stained for SA ‐βGal. * denotes P < 0.05. n = 4. In contrast, increased β‐catenin levels rescued HFA s from senescence induced by meth and HIV (C, D). C). HFA s were transfected with control plasmid pc DNA 3 + , or a plasmid expressing constitutive active β‐catenin (S35Y). Transfected HFA s were treated with 1x PBS vehicle or 300 μ m of meth daily. At day 6, cells were stained for SA ‐βGal. D) HFA s were either infected with VSVG ‐ HIV or mock infected. Next day, cells were washed extensively and cultured in cABM medium in the presence of 5 m m LiCl or control vehicle 1x PBS for 6 days. HFA s were then analyzed for SA ‐βGal expression. * denotes P < 0.05. n = 4.

Article Snippet: Other antibodies include Iba1 antibody (Fisher Scientific #019‐19741, Pittsburgh, PA), active β‐catenin antibody (US Biological C2069‐47, Salem, MA), total β‐catenin antibody (C2206; Sigma), p16 INK4A antibody (Proteintech #22515‐1‐AP, Chicago, IL), GAPDH antibody (G99445; Sigma), antineurofilament heavy chain (Millipore Ab5539, Danvers, MA).

Techniques: Transfection, Control, Western Blot, Staining, Plasmid Preparation, Expressing, Infection, Cell Culture