Journal: bioRxiv

Article Title: Targeting a macrophage stemness factor to mitigate diseases post respiratory viral infection

doi: 10.1101/2025.10.03.680366

Figure Lengend Snippet: A. Representative IF images of TCF4 in Ctnnb1 fl/fl and Ctnnb cKO AM stimulated with or without Poly(I:C). Violin plot representing TCF4 MFI in Ctnnb1 fl/fl or Ctnnb1 cKO AM stimulated with or without Poly(I:C). Scale bar 10 μm. B. Immunoblot of active β-catenin, HIF-1α and β-Actin in Tcf7l2 fl/fl or Tcf7l2 cKO AMs treated with or without Poly(I:C). C. Bar graph showing relative expression of active β-catenin in Tcf7l2 fl/fl or Tcf7l2 cKO AMs treated with or without Poly(I:C) quantified from three individual experiments. D. Bar graph showing relative expression of HIF-1α in Tcf7l2 fl/fl or Tcf7l2 cKO AMs. AMs treated with or without Poly(I:C) quantified from three individual experiments. E. Bar graph showing relative expression of Wnt signaling pathway genes ( Fzd2, Ctnnb1, Wnt1, Wnt4 ) in Tcf7l2 fl/fl or Tcf7l2 cKO AMs treated with or without Poly(I:C). F. Bar graph showing relative expression of inflammatory cytokine expression ( Il1b, Il6, Tnf, and Ccl2 ) in Tcf7l2 fl/fl or Tcf7l2 cKO AMs treated with or without β-catenin inhibitor (XAV939) or HIF-1α inhibitor (LW6). G. Graph showing oxygen consumption rate (OCR) measurements in WT and Tcf7l2 cKO AMs treated with or without β-catenin inhibitor (XAV939) or HIF-1α inhibitor (LW6). Right bar graph of OCR measurements in WT and Tcf7l2 cKO AMs treated with or without β-catenin inhibitor (XAV939) or HIF-1α inhibitor (LW6). Data are representative of at least two independent experiments with similar results, p -values are represented as * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: The following antibodies were used TCF4 (Santa Cruz Biotech sc-166699, 1:200), Ki67 (Thermo, Ki-67, (eBioscienceTM Cat #14-5698-82), Non-phospho (Active) β-Catenin (Cell Signaling Tech Cat#8814 and HIF-1α (Cell Signaling Tech Cat #79233).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Targeting a macrophage stemness factor to mitigate diseases post respiratory viral infection

doi: 10.1101/2025.10.03.680366

Figure Lengend Snippet: A. Representative confocal images of Tcf7l2 fl/fl or Tcf7l2 cKO AM stained with HIF-1α and active β-catenin, stimulated with Poly(I:C) for 24h in vitro . Violin plot showing HIF1α and active β-catenin MFI in WT or Tcf7l2 cKO AM, stimulated with Poly(I:C) for 24h in vitro . Scale bar 10 μm. B. Bar graph showing indicated gene expression of Wnt pathway in sorted GFP + AM transduced with Adv-Control or Adv-Tcf7l2. C. Bar graph showing gene expression of indicated Wnt ligands in sorted GFP + AMs transduced with Adv-Control or Adv-Tcf7l2. D. Bar graph showing gene expression of indicated Wnt ligands in sorted GFP + AMs transduced with Adv-Control or Adv-Tcf7l2. E. Graph showing oxygen consumption rate in Tcf7l2 fl/fl or Tcf7l2 cKO AM treated with HIF-1α and active β-catenin for 24h in vitro . Bar graph showing basal and maximum oxygen consummation rate. F. Bar graph ChIP analysis of β-catenin and TCF4 binding to Enol , and Hk2 in WT AMs treated with Wnt activator. G. Bar graph ChIP analysis of β-catenin and TCF4 binding to Ndufs1 , and Ndufa4 in WT AMs treated with Wnt activator. Data are representative of at least two independent experiments with similar results. p -values are represented as ns = non-significant, * p < 0.05, ** p < 0.01, **** p < 0.001.

Article Snippet: The following antibodies were used TCF4 (Santa Cruz Biotech sc-166699, 1:200), Ki67 (Thermo, Ki-67, (eBioscienceTM Cat #14-5698-82), Non-phospho (Active) β-Catenin (Cell Signaling Tech Cat#8814 and HIF-1α (Cell Signaling Tech Cat #79233).

Techniques: Staining, In Vitro, Gene Expression, Transduction, Control, Binding Assay

Journal: bioRxiv

Article Title: Targeting a macrophage stemness factor to mitigate diseases post respiratory viral infection

doi: 10.1101/2025.10.03.680366

Figure Lengend Snippet: A. Western Blot showing TCF4 expression in WT AMs stimulated with IFN-α and/or IFN-γ for 24h in vitro . B. Representative confocal images in WT AMs stimulated with IFN-α and/or IFN-γ. Right violin plot showing quantification of TCF4 under IFN stimulation. C. Representative confocal images in WT AM stimulated with IFN-α and/or IFN-γ. Right violin plot showing quantification of HIF-1α and active β-catenin under IFN stimulation D. Schematic diagram of the differentiation of purified monocytes from human donors to AM-like macrophages. E. Western blot showing TCF4 expression in monocytes derived AMs from 3 different individuals, donor (D)1-3, under stimulation of IFNγ for 24h. Right graph showing quantification of TCF4 expression under stimulation of IFNγ for 24h. Data are representative of at least two independent experiments with similar results, p -values are represented as ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: The following antibodies were used TCF4 (Santa Cruz Biotech sc-166699, 1:200), Ki67 (Thermo, Ki-67, (eBioscienceTM Cat #14-5698-82), Non-phospho (Active) β-Catenin (Cell Signaling Tech Cat#8814 and HIF-1α (Cell Signaling Tech Cat #79233).

Techniques: Western Blot, Expressing, In Vitro, Purification, Derivative Assay

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: Frontiers in Cell and Developmental Biology

Article Title: Signaling Proteins Recruited to the Sperm Binding Site: Role of β-Catenin and Rho A

doi: 10.3389/fcell.2022.886664

Figure Lengend Snippet: Localization of β-catenin at the sperm binding site.Oocytes fixed at different stages of fertilization were labeled with anti- β-catenin (green) and Alexa Fluor-568 phalloidin (red) as well as Hoechst 33342 (blue). (A) Recently bound sperm. (B) bound sperm with microvilli exhibiting β-catenin labelling. (C) β-catenin plaque colocalizing with the actin layer. (D) sperm undergoing incorporation into the ooplasm with a single bright patch of β-catenin remaining. Images were selected from 30 oocytes recovered from 4 females. Magnification is indicated by the bar.

Article Snippet: Primary antibodies used include rabbit anti-human non-phospho (Active) β−catenin ((Ser33/37/Thr41, Cat # 8814), Cell Signaling, Danvers, MA), rabbit anti- β−catenin (epitope different from the non-phosphorylated antibody from Cell Signaling) [(Cat # BD610153) Becton, Dickinson, and Co. Franklin Lakes, NJ], rabbit anti-human RHOA (Cat. # 10749-1-AP, Proteintech, Rosemont, IL).

Techniques: Binding Assay, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Signaling Proteins Recruited to the Sperm Binding Site: Role of β-Catenin and Rho A

doi: 10.3389/fcell.2022.886664

Figure Lengend Snippet: Distribution of f-actin and β-catenin in the oocyte cortex.MII oocyte from a virgin female was treated by prolonged fixation and labeled with anti- β-catenin (green) and Alexa Fluor-568 phalloidin (red) to label f-actin, as well as Hoechst 33342 (blue). Z-stack images viewed in volume mode (A) , a tangential image (B) , and an equatorial image (C) reveal fenestrae appearing as holes in the cortical actin layer (A,B) or as regions of thinner f-actin in the equatorial view (C) . Images were selected from 10 oocytes from three females. Magnification is indicated by the bar.

Article Snippet: Primary antibodies used include rabbit anti-human non-phospho (Active) β−catenin ((Ser33/37/Thr41, Cat # 8814), Cell Signaling, Danvers, MA), rabbit anti- β−catenin (epitope different from the non-phosphorylated antibody from Cell Signaling) [(Cat # BD610153) Becton, Dickinson, and Co. Franklin Lakes, NJ], rabbit anti-human RHOA (Cat. # 10749-1-AP, Proteintech, Rosemont, IL).

Techniques: Labeling

Journal: Cell Death and Differentiation

Article Title: TRAP1 regulates stemness through Wnt/ β -catenin pathway in human colorectal carcinoma

doi: 10.1038/cdd.2016.67

Figure Lengend Snippet: TRAP1 regulates Wnt/β-catenin pathway in colon carcinoma cells. (a) Real-time PCR gene expression analysis of selected genes in TRAP1-silenced HCT116 and HT29 cells compared with cells transfected with negative siRNA: *P<0.01; **P<0.001. Inserts: TRAP1 immunoblot analysis in control (1) and TRAP1-silenced (2) HCT116 and HT29 cells. Data represent mean + S.D. (b, c) TRAP1, Wnt3A, β-catenin and survivin immunoblot analysis in total cell lysates from HCT116 cells transfected with control and two TRAP1 independent siRNAs (b) and HT29, COLO320 and CaCo2 cells transfected with control or TRAP1 siRNAs (c)

Article Snippet: The following antibodies were used: mouse monoclonal anti-HSP75 (sc-73604), mouse monoclonal anti-PSMC4 (TBP7) (sc-166003), mouse monoclonal anti-Ubiquitin (sc-8017), mouse monoclonal anti-GAPDH (sc-47724) and mouse monoclonal anti-β-Actin (sc-47778) from Santa Cruz Biotechnology, rabbit monoclonal anti-Histone H3 (#4499), rabbit monoclonal anti-active- β -catenin (#8814), rabbit polyclonal anti-phospho- β -catenin (#9561), and rabbit polyclonal anti- β-catenin (#9562), rabbit monoclonal anti-Survivin (#2808) from Cell Signaling Technology (Boston, MA, USA), rabbit monoclonal anti-WNT3A (ab172612) from Abcam (Cambridge, UK), rabbit polyclonal anti-Survivin (NB 500-201H) from Novus Biologicals (Littleton, CO, USA).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Transfection, Western Blot, Control

Journal: Cell Death and Differentiation

Article Title: TRAP1 regulates stemness through Wnt/ β -catenin pathway in human colorectal carcinoma

doi: 10.1038/cdd.2016.67

Figure Lengend Snippet: TRAP1 is responsible for regulation of β-catenin ubiquitination/phosphorylation. (a) Total lysates from HCT116 cells, transfected with control or TBP7 siRNAs, were separated by SDS-PAGE and immunoblotted with indicated antibodies. (b, c) β-catenin immunoprecipitates were obtained from HCT116 cells transfected with TRAP1- or TBP7 siRNAs (b) or with a TBP7 delection mutant (c). Cells were incubated with 10 mmol/l MG132 for 2 h before cell lysis, separated by SDS-PAGE and immunoblotted with the indicated antibodies. Densitometric band intensities represent ratios between ubiquitinated and total β-catenin bands in IPs. Neg Ab, total cellular extracts incubated with non-specific antibody. (c) The arrow indicates the TBP7 deletion mutant band. (d) Total lysates and cytosolic and nuclear fractions were obtained from HCT116 cells transiently silenced for TRAP1 by siRNAs. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with indicated antibodies

Article Snippet: The following antibodies were used: mouse monoclonal anti-HSP75 (sc-73604), mouse monoclonal anti-PSMC4 (TBP7) (sc-166003), mouse monoclonal anti-Ubiquitin (sc-8017), mouse monoclonal anti-GAPDH (sc-47724) and mouse monoclonal anti-β-Actin (sc-47778) from Santa Cruz Biotechnology, rabbit monoclonal anti-Histone H3 (#4499), rabbit monoclonal anti-active- β -catenin (#8814), rabbit polyclonal anti-phospho- β -catenin (#9561), and rabbit polyclonal anti- β-catenin (#9562), rabbit monoclonal anti-Survivin (#2808) from Cell Signaling Technology (Boston, MA, USA), rabbit monoclonal anti-WNT3A (ab172612) from Abcam (Cambridge, UK), rabbit polyclonal anti-Survivin (NB 500-201H) from Novus Biologicals (Littleton, CO, USA).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Transfection, Control, SDS Page, Mutagenesis, Incubation, Lysis

Journal: Cell Death and Differentiation

Article Title: TRAP1 regulates stemness through Wnt/ β -catenin pathway in human colorectal carcinoma

doi: 10.1038/cdd.2016.67

Figure Lengend Snippet: TRAP1 and β-catenin are components of an intracellular protein complex. (a, b) TRAP1 (a) and TBP7 (b) immunoprecipitates were obtained from HCT116 cells, separated by SDS-PAGE and immunoblotted with the indicated antibodies. (c) Representative immunofluorescence images (left panels) and close-up (right panels) showing colocalization of β-catenin (red) with TRAP1 (green). Scale bar, 20 μm. (d) Representative fluorescence image (left panel) and close-up (right panel) showing proximity ligation assay signals (red), detected after labeling HCT116 cells with anti-TRAP1 and anti- β-catenin antibodies. Nuclei were DAPI-labeled (blue). Scale bar, 20 μm. (e) TRAP1 immunoprecipitates were obtained from HCT116 cells transfected with control, TRAP1 or TBP7 siRNAs, separated by SDS-PAGE and immunoblotted with the indicated antibodies. A, B and E. No Ab, Total cellular extracts loaded on Control Agarose Resin without antibody

Article Snippet: The following antibodies were used: mouse monoclonal anti-HSP75 (sc-73604), mouse monoclonal anti-PSMC4 (TBP7) (sc-166003), mouse monoclonal anti-Ubiquitin (sc-8017), mouse monoclonal anti-GAPDH (sc-47724) and mouse monoclonal anti-β-Actin (sc-47778) from Santa Cruz Biotechnology, rabbit monoclonal anti-Histone H3 (#4499), rabbit monoclonal anti-active- β -catenin (#8814), rabbit polyclonal anti-phospho- β -catenin (#9561), and rabbit polyclonal anti- β-catenin (#9562), rabbit monoclonal anti-Survivin (#2808) from Cell Signaling Technology (Boston, MA, USA), rabbit monoclonal anti-WNT3A (ab172612) from Abcam (Cambridge, UK), rabbit polyclonal anti-Survivin (NB 500-201H) from Novus Biologicals (Littleton, CO, USA).

Techniques: SDS Page, Immunofluorescence, Fluorescence, Proximity Ligation Assay, Labeling, Transfection, Control

Journal: Cell Death and Differentiation

Article Title: TRAP1 regulates stemness through Wnt/ β -catenin pathway in human colorectal carcinoma

doi: 10.1038/cdd.2016.67

Figure Lengend Snippet: TRAP1 is co-expressed with β-catenin and Wnt/β-catenin-related genes in human colorectal carcinomas. (a) TRAP1 and β-catenin immunoblot analysis (left panel) and CD166/CD44 expression levels in HCT116 cells transfected with pMock and pTRAP1. (b) Total cell lysates from eight representative human CRCs (T) and respective peritumoral non-infiltrated mucosas (M) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (c) Distribution of TRAP1 and β-catenin protein levels in our series of 59 colorectal carcinomas (P<0.0001). (d) Dot plots representing the correlation between TRAP1 and specific Wnt/β-catenin target genes expression levels obtained from two public data sets of 387 human CRCs

Article Snippet: The following antibodies were used: mouse monoclonal anti-HSP75 (sc-73604), mouse monoclonal anti-PSMC4 (TBP7) (sc-166003), mouse monoclonal anti-Ubiquitin (sc-8017), mouse monoclonal anti-GAPDH (sc-47724) and mouse monoclonal anti-β-Actin (sc-47778) from Santa Cruz Biotechnology, rabbit monoclonal anti-Histone H3 (#4499), rabbit monoclonal anti-active- β -catenin (#8814), rabbit polyclonal anti-phospho- β -catenin (#9561), and rabbit polyclonal anti- β-catenin (#9562), rabbit monoclonal anti-Survivin (#2808) from Cell Signaling Technology (Boston, MA, USA), rabbit monoclonal anti-WNT3A (ab172612) from Abcam (Cambridge, UK), rabbit polyclonal anti-Survivin (NB 500-201H) from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Expressing, Transfection, SDS Page

Journal: Nutrients

Article Title: Selenium Yeast and Fish Oil Combination Diminishes Cancer Stem Cell Traits and Reverses Cisplatin Resistance in A549 Sphere Cells

doi: 10.3390/nu14153232

Figure Lengend Snippet: Floating A549 sphere cells have reduced AMPK activity and elevated markers of CSC and EMT than parental A549 cells. ( A ) Morphologies of adherent parental A549 cells and floating A549 sphere cells after 7 days of culture. Scale bar = 100 μm. Protein levels of ( B ) CSC (CD133 and ABCG2) and EMT (vimentin) markers, anti-apoptotic protein Bcl-2, ( C ) EMT markers (N-cadherin and AXL), oncogenic drivers (active YAP, YAP, and TAZ), ( D ) reported targets of selenium (p-AMPK, β-catenin, and COX-2), and ER stress-response elements (GRP78 and CHOP) in parental A549 cells and A549 sphere cells. Whole-cell lysates were subjected to Western blot analysis for the proteins of interest, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) employed as a loading control. The numbers under the bands indicate the relative densitometric ratios to the bands of the loading control. AMPK, AMP-activated protein kinase; CSC, cancer stem cell; Bcl-2, B-cell lymphoma 2; EMT, epithelial–mesenchymal transition; AXL, anexelekto; YAP, yes-associated protein; TAZ, PDZ-binding motif; p-AMPK, phosphorylated AMPK; COX-2, cyclooxygenase-2; ER, endoplasmic reticulum; CHOP, proapoptotic CCAAT/enhancer-binding protein homologous protein; p-AMPKα, phosphorylated AMPK alpha.

Article Snippet: Primary antibodies against non-phospho (active) β-catenin (Ser33/37/Thr41, #8814), ABCG2 (#4477), phospho-AMPKα (Thr172, #2535), AXL (#8661), TAZ (#8418), active caspase-4 (#4450), full-length caspase-8 (#9746), cleaved caspase-3 (Asp175, #9664), and cleaved caspase-9 (Asp315, #9505) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Activity Assay, Western Blot, Control, Binding Assay

Journal: Nutrients

Article Title: Selenium Yeast and Fish Oil Combination Diminishes Cancer Stem Cell Traits and Reverses Cisplatin Resistance in A549 Sphere Cells

doi: 10.3390/nu14153232

Figure Lengend Snippet: Selenium yeast (Se-Y) and fish oil (FO) combination synergistically induced apoptosis, suppressed cell viability, and modulated ER stress-response elements of A549 sphere cells. ( A ) The apoptotic sub-G1 percentage of A549 sphere cells after treatment with Se-Y, FO alone or in combination for 72 h. ( B ) The percentages of early apoptotic (lower right quadrant, Q1-LR), late apoptotic (upper right quadrant, Q1-UR), necrotic (upper left quadrant, Q1-UL), and viable (lower left quadrant, Q1-LL) A549 sphere cells analyzed by annexin V/7-AAD double staining and flow cytometer. ( C ) Combined effect of Se-Y and FO on the viability (mean ± standard error) of A549 sphere cells after 72 h of treatment (performed in RPMI-1640 medium as described in Materials and Methods section). Combined effects of Se-Y and FO on the protein levels of ( D ) ER stress-response elements (GRP78 and CHOP), COX-2, activated β-catenin, ( E ) caspase-3, 4, 9, full-length caspase-8, and Bcl-2 of A549 sphere cells after 72 h of treatment. Whole-cell lysates were subjected to Western blot analysis for the proteins of interest, employing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The numbers under the bands indicate the relative densitometric ratios to the bands of the loading control.

Article Snippet: Primary antibodies against non-phospho (active) β-catenin (Ser33/37/Thr41, #8814), ABCG2 (#4477), phospho-AMPKα (Thr172, #2535), AXL (#8661), TAZ (#8418), active caspase-4 (#4450), full-length caspase-8 (#9746), cleaved caspase-3 (Asp175, #9664), and cleaved caspase-9 (Asp315, #9505) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Double Staining, Flow Cytometry, Western Blot, Control