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 Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: β-catenin is essential for CUG2-induced CSC-like phenotypes. (A) A549-VEC and A549-CUG2 cells were separated into cytosolic and nuclear fractions. β-catenin expression was determined by performing western blot analysis with an anti-β-catenin antibody. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. The cytosolic and nuclear fractions were confirmed by detecting actin and Sp1, respectively. (B) Total RNAs (3 µ g) were isolated from the A549-VECand A549-CUG2 cells, and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (C) A549-VEC and A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector and were harvested at 48 h after the transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results are an average of three experiments; bars indicate the means ± SD ( *** P<0.001, A549-VEC vs A549-CUG2 cells). (D) Transfection of efficiency of β-catenin siRNAs (#1 or #2) was confirmed by western blot analysis. (E) Migration of A549-CUG2 cells was measured with the wound healing assay at 24 h after control or β-catenin siRNAs (#1 and #2) transfection. Wound closure areas were monitored using a phase-contrast microscope at ×100 magnification, and the assay was repeated twice. (F) Invasion of A549-CUG2 cells transfected with control or β-catenin siRNAs (#1 and #2) was determined using the 48-well Boyden chamber. The chamber was assembled using Matrigel-coated polycarbonate filters. Scale bar indicates 100 µ m, and the assay was repeated twice. Each assay was performed in triplicate, and error bars indicate the means ± SD ( *** P<0.001, control vs. β-catenin siRNA). (G) A549-CUG2 cells (1,000 cells per well) transfected with control or β-catenin siRNAs (#1 and #2) were seeded in ultra-low attachment plates for 2, 4, or 6 days. The assay was performed in triplicate, and error bars indicate the means ± SD ( ** P<0.01 and *** P<0.001, control vs. β-catenin siRNA). Scale bars indicate 50 µ m. CUG2, cancer-upregulated gene 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Migration, Wound Healing Assay, Microscopy

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-induced β-catenin phosphorylation is unique and differs from that induced by Wnt3a. (A) A549-VEC and A549-CUG2 cells were treated with wortmannin (10 µ M), H89 (10 µ M), PP2 (2.5 µ M) and DMSO (mock) in the presence of Wnt3a (100 ng/ml) or PBS as a vehicle for 12 h, and cell lysates were prepared. Proteins present in the cell lysates were separated by performing SDS-PAGE on 10% gels, and β-catenin and α-actin were detected using corresponding antibodies. (B and C) Proteins present in the lysates of A549-VEC cells treated with Wnt3a (100 ng/ml) or PBS, and of A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were determined using antibodies against phosphorylated Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (D) Lysates of A549-VEC and A549-CUG2 cells treated with a control or GSK3β siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. GSK3β and phosphorylated GSK3β were detected by performing western blot with corresponding antibodies. CUG2, cancer-upregulated gene 2; GSK3β, glycogen synthase kinase 3β.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Phospho-proteomics, SDS Page, Control, Western Blot

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-activated NEK2 is responsible for the phosphorylation of β-catenin at Ser33/Ser37. (A) Lysates of A549-VEC and A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels, and NEK2 was detected by performing western blot analysis with anti-NEK2 antibody. (B) Levels of NEK2 and β-catenin phosphorylated at Ser33/Ser37 (p-β-catenin; p-Ser33/Ser37) in A549-VEC and A549-CUG2 cells were determined by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) β-catenin was pulled down from the lysates of A549-VEC and A549-CUG2 cells by using an anti-β-catenin antibody. NEK2 present in the immunoprecipitates was detected using an anti-NEK2 antibody, and β-catenin present in the immunoprecipitates was detected as a loading control by using an anti-β-catenin antibody. (D) NEK2 was pulled down from A549-VEC and A549-CUG2 cells by using an anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay, including immunoprecipitates as NEK2 kinase, recombinant GST-β-catenin as a substrate, ATP, and a reaction buffer, was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by determining the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing immunoblotting, and β-catenin levels were examined as a loading control. (E) Lysates of A549-CUG2 cells transfected with the control or NEK2 siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (F) Following transfection with control or NEK2 siRNA#1, the levels of p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells were detected by performing immunofluorescence microscopy with Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Recombinant, Incubation, Activity Assay, Transfection

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment destabilizes β-catenin in a proteasome- and PKCα- dependent manner. (A) A549-CUG2 cells were treated with CGK062 (0, 1, 10, 50 and 100 µ M) for 24 h, and β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (B) A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector in the presence of CGK062 (50 µ M) and were harvested at 48 h following transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results shown are an average of 3 experiments; bars indicate the means ± SD ( *** P<0.001, 0 vs. 10 µ M, 0 vs. 50 µ M). (C) Total RNAs (3 µ g) were isolated from A549-CUG2 cells treated with or without CGK062 (50 µ M), and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (D) A549-CUG2 cells were treated with MG132 (1 µ M) for 8 h in the absence or presence of CGK062 (50 µ M) and were harvested. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (E) A549-CUG2 cells were treated with BMI (7.5 µ M), a specific PKCα inhibitor, or were transfected with a control and PKCα siRNAs (#1 or #2) in the absence or presence of CGK062 (50 µ M) for 24 h. Transfection efficiency of PKCα siRNAs (#1 or #2) was confirmed by western blot analysis. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. CUG2, cancer-upregulated gene 2; PKC, protein kinase C.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment decreases NEK2 expression and kinase activity in A549-CUG2 cells. (A) Lysates of A549-CUG2 cells treated with CGK062 (0, 10, 30, 40 and 50 µ M) for 24 h were separated by performing SDS-PAGE on 10% gels. The expression of NEK2, β-catenin, E-cadherin, N-cadherin and vimentin was detected by performing western blot analysis with corresponding antibodies. (B) Levels of NEK2 and p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO as Mock were detected by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) NEK2 was pulled down from A549-VEC and A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO by using the anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by detecting the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing western blot analysis, and β-catenin levels were examined as a loading control. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Antibodies against Smad2/3 (#5678), phosphorylated Smad2 (#3108), β-catenin (#9562), and phosphorylated β-catenin (Ser33/Ser37/Thr41, Ser45, or Ser675; #9561, #9564, or #4176, respectively) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Activity Assay, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Incubation, Phospho-proteomics

Journal: PLoS ONE

Article Title: Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway

doi: 10.1371/journal.pone.0147018

Figure Lengend Snippet: HK-2 cells were pretreated with NaHS at the indicated doses for 12 hours and then they were treated by TGF-β1 for the following 36 hours. (A) Western blot assay for β-catenin and non-phosphorylated β-catenin expression in HK-2 cells. (B) Graphical representation of the relative quantification for β-catenin and non-phosphorylated β-catenin. The relative values were calculated by the density of β-catenin and non-phosphorylated β-catenin vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. # P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The primary antibody for non-phosphorylated β-catenin (1:500, Cell Signaling Technology, USA) was incubated overnight at 4°C.

Techniques: Western Blot, Expressing, Quantitative Proteomics, Control, Comparison

Journal: PLoS ONE

Article Title: Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway

doi: 10.1371/journal.pone.0147018

Figure Lengend Snippet: (A,B,C,D) HK-2 cells were pretreated with or without NaHS (100μM) for 12 hours and then they were treated by TGF-β1 for the following 36 hours. Blue color stands for nucleus stained by DAPI and green color stands for non-phosphorylated β-catenin. (E) Graphical representation of the cells in which non-phosphorylated β-catenin translocated into nucleus. The values of mean ± SEM (n = 3) were gained from three randomly microscope fields in each slide. # P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The primary antibody for non-phosphorylated β-catenin (1:500, Cell Signaling Technology, USA) was incubated overnight at 4°C.

Techniques: Staining, Microscopy, Control, Comparison

Journal: PLoS ONE

Article Title: Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway

doi: 10.1371/journal.pone.0147018

Figure Lengend Snippet: (A) The expression of non-phosphorylated β-catenin under the condition of ERK signal was blocked. ERK1/2 inhibitor U0126 (10μM) was given to HK-2 cells for 1 hour and then cells were incubated with TGF-β1 for 36 hours. (B) Effects of NaHS (100μM) on the expression of fibronectin, E-cadherin,TβR I and α-SMA in the presence of TGF-β1 and U0126 or the β-catenin inhibitors XAV-939. Pretreatment by NaHS (100μM) is given to HK-2 cells for 12 hours and then U0126 (10μM) or XAV-939 (10μM) were given to the cells for 1 hour before they were incubated with TGF-β1 for the following 36 hours. (C) Graphical representation of the relative quantification for the protein level. The relative values were calculated by the density of targeted proteins vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. # P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The primary antibody for non-phosphorylated β-catenin (1:500, Cell Signaling Technology, USA) was incubated overnight at 4°C.

Techniques: Expressing, Incubation, Quantitative Proteomics, Control, Comparison

Journal: PLoS ONE

Article Title: Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway

doi: 10.1371/journal.pone.0147018

Figure Lengend Snippet: (A) β-catenin knockdown efficiency by siRNA in HK-2 cells. The cells were transfected with β-catenin siRNA or control siRNA for 6 hours and the culture medium was replaced by fresh medium. Western blot assay was used to detect the expression level of non-phosphorylated β-catenin. (B) After transfected with β-catenin siRNA or control siRNA for 6 hours, HK-2 cells were incubated with NaHS (100μM) for 12 hours and then with TGF-β1 for the following 36 hours. The expression level of fibronectin, E-cadherin, TβR I and α-SMA were examined by western blot assay. (C) Graphical representation of the relative quantification for the protein level. The relative values were calculated by the density of targeted proteins vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. # P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The primary antibody for non-phosphorylated β-catenin (1:500, Cell Signaling Technology, USA) was incubated overnight at 4°C.

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Incubation, Quantitative Proteomics, Comparison

Journal: Theranostics

Article Title: Homozygous MESP1 knock-in reporter hESCs facilitated cardiovascular cell differentiation and myocardial infarction repair

doi: 10.7150/thno.42347

Figure Lengend Snippet: MESP1 represses canonical Wnt/β-CATENIN signaling during hESC cardiac differentiation. ( A ) Dox inducible MESP1 expression system. Left, dosage effect of Dox on MESP1-Flag protein expression (Dox concentration is indicated at the top). Middle and right, MESP1-Flag protein was induced 3 h after Dox addition and disappeared 6 h after Dox withdraw. ACTIN was used as the loading control in all experiments. ( B ) Schematic view of the differentiation protocol. Cells were treated with 25 ng/mL BMP4 and 2 µM CHIR99021 for 24 h. 48 h after BMP4 and CHIR99021 removal, 5 µM IWP2 or 400 ng/mL Dox was added for 48 h. After IWP2 or Dox treatment, cells were cultured in RPMI/B27 minus insulin medium until day 7, then changed to RPMI/B27 medium until day 12. ( C ) Bar graph showing cTnT + cell percentage in untreated, Dox, or/and IWP2 treated cells based on flow cytometry analysis ( n = 3). Data represent mean ± SEM. ( D ) Q-PCR analysis of genes induced by MESP1. Dox or IWP2 were added during day 3-5. The expression levels of cardiac and Wnt pathway genes were normalized against GAPDH. The expression levels in untreated cells were set as “1”. ( n = 3). ( E ) Western blot showing MESP1, phosphorylated β-CATENIN (phos-β-CATENIN), and total β-CATENIN protein levels in cells treated IWP2 or Dox, ACTIN was used as the loading control ( n = 3). ( F ) Schematic view of WNT5A promoter region. Dark blue boxes indicated E-box motifs. Red lines indicate regions tested in ChIP-Q-PCR assay. TSS: transcription start site. ( G ) Timeline of ChIP-Q-PCR analysis. ( H ) ChIP-Q-PCR analysis of MESP1 binding to E-box motifs on the WNT5A promoter. A non-related genomic region was used as negative control. Results were normalized against the enrichment of the same DNA fragment without Dox induction. A, B, C and D are E-box regions depicted in (F). ( I ) Luciferase reporter assay showing the transcription activity of E-box A ( WNT5A promoter); mut E-box A ( WNT5A promoter with E-box A mutated); ΔE-box A (E-box A deleted WNT5A promoter). Empty vector was used as negative control. Data represent the mean ± SEM, n = 3, * p < 0.05, ** p < 0.005, *** p < 0.001, Student's t -test. ( J ) Western blot showing induction of WNT5A in hESCs reduced the level of total β-CATENIN protein. GAPDH, loading control. ( K ) Schematic view of differentiation protocol, cells were treated with WNT5A instead of IWP2 during day 3-5. ( L ) FACS analysis of cTnT on day 12 of differentiation.

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBST and then incubated with primary antibodies against MESP1 (APR39374_P050, AVIVA Systems Biology), Flag-tag (M2, Sigma), β-CATENIN (Cell signaling technology), phosphorylated-β-CATENIN (Bioworld) and ACTIN (Proteintech).

Techniques: Expressing, Concentration Assay, Cell Culture, Flow Cytometry, Western Blot, Binding Assay, Negative Control, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling

doi: 10.1101/2020.03.09.982231

Figure Lengend Snippet: Merged representative immunofluorescent images of non-phosphorylated-β-catenin (non-p-β-catenin) and Dapi costaining ( A ) and quantification ( B ) of non-p-β-catenin of primary hMSCs cultured on NX-MC or MC for 3 or 7 days. Western blot of primary hMSCs cultured on NX-MC or MC for 0, 3, 7, or 14 days in osteogenic differentiation medium for ( C ) non-p-β-catenin, phosphorylated Akt (p-Akt), and total Akt. Densitometric quantification of western blot analyses demonstrating relative protein amounts of non-p-β-catenin to actin ( D ) and p-Akt to total Akt ( E ). QPCR of primary hMSCs cultured on NX-MC or MC for 3 or 7 days in osteogenic differentiation medium for the ( F ) WNT3A, ( G ) WNT4, ( H ) WNT5A, ( I ) WNT5B, and ( J ) WNT11 (n=3). Bars represent means, errors bars represent SE. Significant posthoc comparisons following ANOVA indicated with p values.

Article Snippet: Western analysis was carried out with primary antibodies against phosphorylated Smad1/5 (p-Smad1/5), total Smad5, phosphorylated Akt (p-Akt), total Akt, phosphorylated FAK (p-FAK), total FAK, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, OPG, RUNX2, YAP, TAZ, non-phosphorylated-β-catenin (non-p-β-catenin), GAPDH, and β-actin and the respective species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling

doi: 10.1101/2020.03.09.982231

Figure Lengend Snippet: Western blot of primary hMSCs cultured on NX-MC or MC for 0 or 3 days in osteogenic differentiation medium untreated, treated with 0.5 μM of LA, or treated with 3 μg/mL of C3 for ( A ) Runx2, OPG, non-p-β-catenin, and GAPDH or ( D ) p-Smad1/5 and Smad5. Densitometric quantification of western blot analyses demonstrating relative protein amounts of ( B ) Runx2 to GAPDH, ( C ) non-p-β-catenin to GAPDH, or ( E ) p-Smad1/5 to Smad5. QPCR of primary hMSCs cultured on NX-MC or MC for 0 or 3 days in osteogenic differentiation medium untreated, treated with 0.5 μM of LA, or treated with 3 μg/mL of C3 for ( F ) BMP2, ( G ) BMP4, and ( H ) BMP7. Bars represent means, errors bars represent SE. Significant posthoc comparisons following ANOVA indicated with p values.

Article Snippet: Western analysis was carried out with primary antibodies against phosphorylated Smad1/5 (p-Smad1/5), total Smad5, phosphorylated Akt (p-Akt), total Akt, phosphorylated FAK (p-FAK), total FAK, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, OPG, RUNX2, YAP, TAZ, non-phosphorylated-β-catenin (non-p-β-catenin), GAPDH, and β-actin and the respective species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling

doi: 10.1101/2020.03.09.982231

Figure Lengend Snippet: Mechanical signals, such as stiffness, from MC-GAG contribute to inducing differentiation of osteoprogenitors in a manner that requires activation of YAP/TAZ (1A) resulting in the activation of β-catenin (1B). In contrast, osteogenic differentiation downstream of activation of the canonical BMPR signaling pathway occurs in a YAP/TAZ-independent manner on MC-GAG (2), suggesting that this pathway is activated via a non-mechanical trigger.

Article Snippet: Western analysis was carried out with primary antibodies against phosphorylated Smad1/5 (p-Smad1/5), total Smad5, phosphorylated Akt (p-Akt), total Akt, phosphorylated FAK (p-FAK), total FAK, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, OPG, RUNX2, YAP, TAZ, non-phosphorylated-β-catenin (non-p-β-catenin), GAPDH, and β-actin and the respective species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: 4-Aminoethylamino-emodin – a novel potent inhibitor of GSK-3β– acts as an insulin-sensitizer avoiding downstream effects of activated β-catenin

doi: 10.1111/j.1582-4934.2009.00701.x

Figure Lengend Snippet: Influence of different GSK-3β inhibitors on total amount and activation of β-catenin. (A) Dose-dependent accumulation of activated and (B) total β-catenin in HEK293. Twenty-four hours before stimulation 0.5 × 10 6 HEK293 cells per well were seeded into six-well plates. To inhibit GSK-3β, cells were treated with different concentrations of LiCl (10, 20, 40, 80 and 160 mM; lanes 3–12), SB216763 (2.5, 5, 10, 20 and 40 μM; lanes 3–12) and L4 (2.5, 10, 40, 80 and 160 μM; lanes 3–12). Controls were either untreated (LiCl; lanes 1, 2) or received DMSO at a final concentration of 0.8% (SB216763 and L4; lanes 1, 2). Cells were harvested 3 hrs after adding GSK-3 β inhibitors and cell lysates were prepared. After determination of protein concentrations and the addition of GST-β-catenin calibrator proteins, duplicate samples of each cell lysate were separated by SDS-PAGE, and subsequently transferred onto nitrocellulose. Western blots were sequentially probed with antibodies recognizing (A) active β-catenin (anti-ABC), (B) total cellular β-catenin and (C) α-tubulin. (D) Affinity precipitation to measure a signalling-competent pool of β-catenin induced by Wnt-3a or GSK-3β inhibitors. Twenty-four hours before stimulation 5 × 10 6 murine C17.2 cells were seeded into 100 mm plates. To induce a free pool of β-catenin cells were treated with SB216763 (20 μM), L4 (40 μM) or Wnt-3a-conditioned media (Wnt-3a CM). Controls were either untreated, received DMSO at a final concentration of 0.4% or were incubated with mock conditioned media (cont. CM). Cells were harvested 3 hrs after adding GSK-3β inhibitors or Wnt-3a CM and cell lysates were prepared. To recover the pool of free cytosolic β-catenin, samples of each cell lysate were supplemented with glutathione sepharose beads and either GST or the GST-ECT fusion protein as indicated. After the binding reaction and extensive washing, proteins bound to the glutathione sepharose matrix were eluted with SDS-PAGE loading buffer, separated by SDS-PAGE and analysed by Western blotting with an antibody recognizing β-catenin. Lanes 1, 2: untreated control cells; lanes 3, 4: DMSO-treated cells; lanes 5, 6: cells treated with mock-conditioned media; lanes 7, 8: SB216763-treated cells; lanes 9, 10: L4-treated cells; lanes 11, 12: cells treated with Wnt-3a-conditioned media. (E) In parallel, a sample of each lysate used for GST-ECT fishing was combined with a mixture with 2.5 ng of GST-fusion proteins carrying amino acid residues 400–781 and 536–781 of β-catenin and total levels of β-catenin were analysed by Western blotting with anti-β-catenin and anti-α-tubulin antibodies. MW: molecular weight marker.

Article Snippet: Alternatively, phosphorylated β-catenin was detected by the phospho-β-catenin (Ser33/37/Thr41) antibody (Cell Signaling Technology, Inc.) in Western Blots.

Techniques: Activation Assay, Concentration Assay, SDS Page, Western Blot, Affinity Precipitation, Incubation, Binding Assay, Control, Molecular Weight, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: 4-Aminoethylamino-emodin – a novel potent inhibitor of GSK-3β– acts as an insulin-sensitizer avoiding downstream effects of activated β-catenin

doi: 10.1111/j.1582-4934.2009.00701.x

Figure Lengend Snippet: Influence of GSK-3β inhibiting agents Wnt-3a, SB216763, L4 and combinations thereof on (A) phosphorylation of glycogen synthase and β-catenin and (B) a signalling-competent pool of β-catenin in primary mouse hepatocytes. Twenty-four hours before stimulation 2 × 10 6 primary murine hepatocytes were seeded into 60 mm plates. To induce phosphorylation events at glycogen synthase and β-catenin or a free pool of β-catenin cells were treated with 200 ng/ml recombinant Wnt-3a (rWnt-3a), 80 μM SB216763, increasing concentrations of L4, or combinations of these reagents as indicated. Control cells were left either untreated or received DMSO at a final concentration of 0.4%. Cells were harvested 3 hrs after adding Wnt-3a or the GSK-3 inhibitors, and cell lysates were prepared as in . Part of this lysate was used (A) for Western Blots using phospho-glycogen synthase (Ser641) antibody, glycogen synthase (15B1) Rabbit mAb, and phospho-β-catenin (Ser33/37/Thr41) antibody or (B) for GST-ECT affinity precipitations as described in using GST-β-catenin fusions proteins as normalizers as indicated. Western blotting signals were quantified and processed as described in ‘Materials and methods’ and in . The results shown in (A) represent average values of phospho-glycogen synthase normalized to total glycogen synthase and those shown in (B) represent average values of normalized β-catenin amounts derived from the same three independent experiments. The corresponding standard errors are given. The results are expressed as (A) percentage of control cells (100%) or as (B) fold changes of β-catenin amounts relative to untreated control cells (=1).

Article Snippet: Alternatively, phosphorylated β-catenin was detected by the phospho-β-catenin (Ser33/37/Thr41) antibody (Cell Signaling Technology, Inc.) in Western Blots.

Techniques: Phospho-proteomics, Recombinant, Control, Concentration Assay, Western Blot, Derivative Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: 4-Aminoethylamino-emodin – a novel potent inhibitor of GSK-3β– acts as an insulin-sensitizer avoiding downstream effects of activated β-catenin

doi: 10.1111/j.1582-4934.2009.00701.x

Figure Lengend Snippet: Scheme illustrating the differential influence of the novel GSK-3β inhibitor L4 and of established inhibitors, SB216763, TDZD-8 and LiCl on metabolic events and β-catenin-dependent gene expression. Established inhibitors (SB216763, TDZD-8 and LiCl) inhibit GSK-3β irrespective of whether it is bound to Axin or not resulting in activation of metabolic events ( e.g. glycogen synthesis) as well as β-catenin-dependent gene expression. The novel inhibitor L4 has a similar influence on GSK-3β, but additionally inhibits an event downstream to dephosphorylation of β-catenin. Thus, L4 favours activation of metabolic events, while avoiding β-catenin-dependent gene expression.

Article Snippet: Alternatively, phosphorylated β-catenin was detected by the phospho-β-catenin (Ser33/37/Thr41) antibody (Cell Signaling Technology, Inc.) in Western Blots.

Techniques: Gene Expression, Activation Assay, De-Phosphorylation Assay

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-934 promotes colorectal cancer cell proliferation by directly targeting Dickkopf-related protein 2

doi: 10.3892/etm.2021.10473

Figure Lengend Snippet: Sequences of primers used for quantitative PCR.

Article Snippet: β-catenin (cat. no. 8480; 1:2,000) and phosphorylated (p)-β-catenin (Ser675; cat. no. 4176; 1:2,000) antibodies were purchased from Cell Signaling Technology, Inc. GAPDH (cat. no. ab8245; 1:5,000) antibody was purchased from Abcam. c-Myc (cat. no. sc40; 1:2,000) and cyclin D1 (cat. no. sc450; 1:2,000) antibodies were purchased from Santa Cruz Biotechnology, Inc. HRP-conjugated secondary antibodies against mouse (cat. no. ab205719; 1:10,000) and rabbit (cat. no. ab6721; 1:10,000) were purchased from Abcam.

Techniques: Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-934 promotes colorectal cancer cell proliferation by directly targeting Dickkopf-related protein 2

doi: 10.3892/etm.2021.10473

Figure Lengend Snippet: miR-934 downregulation inhibits Wnt signaling in HCT116 cells. (A) Western blotting showed that miR-934 downregulation decreased p-β-catenin protein levels in HCT116 cells. (B) TOP/FOP reporter assay was performed in HCT116 cells. The data showed that downregulation of miR-934 repressed the reporter activity in the cells. Downregulation of miR-934 decreased c-Myc and cyclinD1 (C) mRNA and (D) protein expression in HCT116 cells. Pearson's correlation analysis suggested that there was a negative correlation (E) between miR-934 and c-Myc mRNA expression and (F) between miR-934 and cyclinD1 mRNA expression in the collected tumor tissue samples. ** P<0.01 and *** P<0.001 vs. the miR-NC inhibitor group. miR, microRNA; NC, negative control; p, phosphorylated.

Article Snippet: β-catenin (cat. no. 8480; 1:2,000) and phosphorylated (p)-β-catenin (Ser675; cat. no. 4176; 1:2,000) antibodies were purchased from Cell Signaling Technology, Inc. GAPDH (cat. no. ab8245; 1:5,000) antibody was purchased from Abcam. c-Myc (cat. no. sc40; 1:2,000) and cyclin D1 (cat. no. sc450; 1:2,000) antibodies were purchased from Santa Cruz Biotechnology, Inc. HRP-conjugated secondary antibodies against mouse (cat. no. ab205719; 1:10,000) and rabbit (cat. no. ab6721; 1:10,000) were purchased from Abcam.

Techniques: Western Blot, Reporter Assay, Activity Assay, Expressing, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-934 promotes colorectal cancer cell proliferation by directly targeting Dickkopf-related protein 2

doi: 10.3892/etm.2021.10473

Figure Lengend Snippet: miR-934 regulates Wnt/β-catenin signaling by targeting DKK2. (A) Transfection of DKK2 siRNA decreased the DKK2 protein expression level in HCT116 cells. *** P<0.001 vs. control siRNA. (B) DKK2 knockdown reversed downregulation of p-β-catenin protein level induced by miR-934 inhibitor in HCT116 cells. *** P<0.001 vs. miR-NC inhibitor + control siRNA and ## P<0.01 vs. miR-934 inhibitor + control siRNA. (C) DKK2 knockdown reversed the downregulation of c-Myc and cyclinD1 mRNA expression levels induced by miR-934 inhibitor in HCT116 cells. *** P<0.001 vs. miR-NC inhibitor + control siRNA; ### P<0.001 vs. miR-934 inhibitor + control siRNA. siRNA, small interfering RNA; miR, microRNA; NC, negative control; DDK2, Dickkopf-related protein 2.

Article Snippet: β-catenin (cat. no. 8480; 1:2,000) and phosphorylated (p)-β-catenin (Ser675; cat. no. 4176; 1:2,000) antibodies were purchased from Cell Signaling Technology, Inc. GAPDH (cat. no. ab8245; 1:5,000) antibody was purchased from Abcam. c-Myc (cat. no. sc40; 1:2,000) and cyclin D1 (cat. no. sc450; 1:2,000) antibodies were purchased from Santa Cruz Biotechnology, Inc. HRP-conjugated secondary antibodies against mouse (cat. no. ab205719; 1:10,000) and rabbit (cat. no. ab6721; 1:10,000) were purchased from Abcam.

Techniques: Transfection, Expressing, Control, Knockdown, Small Interfering RNA, Negative Control