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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: TGF-β2 antisense oligonucleotide enhances T-cell mediated anti-tumor activities by IL-2 via attenuation of fibrotic reaction in a humanized mouse model of pancreatic ductal adenocarcinoma.

doi: 10.1016/j.biopha.2022.114212

Figure Lengend Snippet: Fig. 4. TASO reduces active β-catenin and related fibrosis in xenografted AsPC-1 tumors. Active β-catenin, α-SMA expression, and collagen synthesis in the tumor were measured by IHC or Sirius red staining. (A) Representative images of Sirius red staining (Bar = 200 µm), IHC for active β-catenin (Bar = 20 µm) and p-SMAD2/3 (Bar = 50 µm) in tumors and their relative intensities were observed (B, C, D). Active β-catenin and α-SMA expression in the tumor was confirmed by Western blot analysis. (E) Representative band images of proteins and the relative intensity ratio of active β-catenin/β-actin (F) and α-SMA/β-actin (G) were presented. IHC data were evaluated in at least four fields per specimen and four to five specimens per group and Western blot data are obtained from four mice per group. Data are expressed as means ± S.E.M. *p < 0.05 and **p < 0.01 vs. PBMC+vehicle group; #p < 0.05 and ##p < 0.01 vs. PBMC+IL-2 group (Dunnet’s test).

Article Snippet: Tissue sections were reacted overnight with rabbit monoclonal primary antibodies against active β-catenin (clone D13A1; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:400), α-smooth muscle actin (α-SMA; clone D4K9N; Cell Signaling Technology, Inc.; 1:100), human CD8 (clone D8A8Y; Cell Signaling Technology, Inc.; 1:200), granzyme B (clone D6E9W; Cell Signaling Technology, Inc.; 1:200), human Foxp3 (clone D2W8E; Cell Signaling Technology, Inc.; 1:200), human programmed death-ligand 1 (PD-L1; clone 28–8; Abcam, Cambridge, UK; 1:400), and fibroblast activation protein-α (FAP-α; clone EPR20021; Abcam; 1:200) and polyclonal antibodies against TGF-β2 (Abcam, 1:100), p-SMAD2/3 (Thermo Fisher Scientific, Waltham, MA, USA; 1:200), and cleaved caspase-3 (Cell Signaling Technology, Inc.; 1:400).

Techniques: Expressing, Staining, Western Blot

Journal: Oncotarget

Article Title: Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth

doi: 10.18632/oncotarget.12650

Figure Lengend Snippet: A . Expression of β-catenin target genes in Capan-2 cells after treatment with DMSO or 10 μM BAY ACC002 for 72 h. RNA was extracted and the mRNA expression level of β-catenin target genes was determined by qRT-PCR. B . Capan-2 cells were treated 200ng recombinant human WNT3A, and grown in the presence of DMSO or 10 μM BAY ACC002 for 72 h. RNA was extracted and the mRNA expression level of AXIN2 was determined by qRT-PCR. C . HEK293-TOP cells were stimulated with 200ng recombinant human WNT3A, and grown in the presence of DMSO or 10 μM BAY ACC002 for 72 h. RNA was extracted and the mRNA expression level of AXIN2 was determined by qRT-PCR. D . Expression of HH target genes in Capan-2 cells, treated with BAY ACC002. Cells were treated as in (A) and mRNA expression levels were determined by qRT-PCR. E . Expression of AXIN2 and GLI1 in Capan-2 cells treated with DMSO or 10 μM BAY ACC002 for 96 h. The cells were then lysed and protein levels were detected by Western blot. GAPDH levels were monitored as a control. F . Growth curves of DanG cells, treated with varying concentrations of BAY ACC002 (arrow indicates time of drug addition). Cell growth was measured over time using the xCELLigence system. Experiments were performed in triplicate. G ., H . and I . Capan-2, BxPC-3 and Panc-1 cells were treated with BAY ACC002, and cell growth over time was determined as in (G). Each bar in (A) - (D) represents mean±SEM ( n = 3-4 for (A), n = 2-3 for (B)-(D), *, p < 0.05, **, p < 0.01, ***, p < 0.001, Student's t test).

Article Snippet: SHH (C9C5) Rabbit monoclonal antibody (2207), non-phospho β-catenin (Ser33/37/Thr41) (D13A1) rabbit monoclonal antibody (8814), and AXIN2 (76G6) rabbit monoclonal antibody (2151) were purchased from Cell Signaling (Danvers, MA, US).

Techniques: Expressing, Quantitative RT-PCR, Recombinant, Western Blot, Control

Journal: Oncotarget

Article Title: Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth

doi: 10.18632/oncotarget.12650

Figure Lengend Snippet: A . AXIN2 and GLI1 expression in Capan-2 pancreatic cancer tumors, treated with vehicle or 30 mg/kg/day BAY ACC002 for 7 days. RNA was extracted from the tumors and expression of AXIN2 and GLI1 was determined by qRT-PCR. Each bar represents mean±SEM, with individual animals represented by dots ( n = 4, *, p < 0.05). B . IHC analysis of β-catenin expression in representative pancreatic cancer sections from mice, carrying Capan-2 or PAXF 2046 tumors, treated with vehicle or BAY ACC002 (35 mg/kg/day on days 1-5, and then 30 mg/kg/day on a 3 days ON/1 day OFF schedule until day 35 for the Capan-2 model; and 30 mg/kg/day for 29 days for the PAXF 2046 model). Arrows point to nuclear β-catenin expression. C . IHC analysis of GLI1 expression in representative pancreatic cancer sections from Capan-2 and PAXF 2046 tumors described in (B). Arrows point to GLI1 positive cells. D . H&E, Alcian blue, and E-cadherin IHC staining of representative sections from the PAXF 2046 tumors, described in (B). Scale bar for (B), (C), and the third set of (D) is 50 μm, and for the first two sets of (D), 100 μm.

Article Snippet: SHH (C9C5) Rabbit monoclonal antibody (2207), non-phospho β-catenin (Ser33/37/Thr41) (D13A1) rabbit monoclonal antibody (8814), and AXIN2 (76G6) rabbit monoclonal antibody (2151) were purchased from Cell Signaling (Danvers, MA, US).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry

Journal: bioRxiv

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.1101/594184

Figure Lengend Snippet: (A) Immunoblotting of KMF, KT3 and KT13 cell lysates for FAK, Pyk2, β-catenin, and actin. (B) KMF and FAK-/- (clone KT13) cell viability treated with DMSO (control), CP (1 μM), JQ1 (1 μM), MLN8237 (1 μM), or MSAB (10 μM) after 72 h as measured by Alamar Blue. Values are means (± SEM, * P<0.05, ** P <0.01, *** P <0.001, one-way ANOVA with Fisher’s LSD multiple comparison test) for 3 independent experiments. (C) Immunoblotting for pY397 FAK, FAK, and actin in lysates of KMF, FAK-/- KT13, GFP-FAK-WT, and GFP-FAK R454 re-expressing cells. (D) Top ten proteomic differences (fold-change) detected by mass spectroscopy of membrane associated proteins in FAK-/- KT13, GFP-FAK-WT, and GFP-FAK R454 re-expressing cells. (E) Immunoblotting for active β-catenin, β-catenin, and actin in lysates of FAK-/- KT13 cells stably expressing GFP-FAK WT, GFP-FAK R454, or stabilized β-catenin (ΔGSK). (F) Growth of the indicated KMF, FAK-/- KT13, or the indicated reconstituted cells in PromoCell XF for 5 days. Values are means (± SEM, * P <0.05, one-way ANOVA) from 2 independent experiments. (G) Tumorsphere formation in PromoCell XF (5 days). (H) Representative images of the indicated cells in tumorsphere formation assays. Scale is 1 mm. (I) ALDEFLUOR activity. Values are means (± SEM **P<0.01, *** P <0.001, one-way ANOVA with a Tukey’s multiple comparisons test) of 3 (panel G) or 4 (panel I) independent experiments. (J) Immunoblotting for ALDH-1A2, ALDH-1B1, Myc, and actin in the indicated cell lysates. (K) Viability (Alamar Blue) of KMF (black circles), KT13 FAK-/- (red squares), GFP-FAK WT (green triangle), GFP-FAK R454 (magenta triangle), and ΔGSK β-catenin (brown diamond) expressing cells treated with increasing CP concentrations for 5 days. Values are means (± SEM, **, P <0.01, *** P <0.001, two-way ANOVA with a Bonferroni’s multiple comparisons test) from 3 independent experiments. EC50 values were determined independently and calculated using Prism (Graphpad).

Article Snippet: Monoclonal antibodies were used to detect E-cadherin (Cell Signaling Technologies [CST] clone 4A2), β-actin (Sigma clone AC-74 or Proteintech clone 7D2C10), β-catenin (CST clone D10A8), non-phospho (active) β-catenin (Ser33/Ser37/Thr41) (CST clone D13A1), Myc (CST clone D84C12), Pyk2 (CST clone 5E2), p21CIP1 (Santa Cruz Biotechnology [SCB}, clone F5), GFP (SCB clone B2), p53 (SCB Pab 240), and α-tubulin (Sigma clone DM1A).

Techniques: Western Blot, Expressing, Mass Spectrometry, Stable Transfection, Activity Assay

Journal: Frontiers in Physiology

Article Title: Using Imaging Mass Cytometry to Define Cell Identities and Interactions in Human Tissues

doi: 10.3389/fphys.2021.817181

Figure Lengend Snippet: Representative Kidney-MAPPS analysis of healthy human kidney tissue for identification of resident cell populations. (A) Pseudocolored image of a region of interest analyzed by IMC. The depicted colors correspond to the markers listed on the image. AQP1, aquaporin-1; UMOD, uromodulin; bCatenin, β-Catenin; CK7, cytokeratin-7; and ERG, ETS-related gene. (B,C) Probability images for nuclei (B) and tubular cells (C) , created using the Ilastik software. (D) Segmentation mask generated in CellProfiler overlaid on a pseudocolored image created in HistoCAT. Highlighted markers were selected in order to represent the tissue structure analogous to (A) while allowing visibility of the cell segmentation. (E) tSNE plot showing different cell types/clusters identified through PhenoGraph analysis of three representative healthy kidney samples in HistoCAT. PT, proximal tubule; DCT, distal convoluted tubule; TAL, thick ascending limb; Podo, podocytes; CD, collecting duct; Endo, endothelial cells (non-glomerular); and Endo(G), glomerular endothelial cells. (F,G) Quantification of the cell types identified in (E) as a percentage of the total number of cells (F) and the number of cells per mm 2 area (G) . Bars show the mean ± SD. n = 3.

Article Snippet: β-Catenin , Tubular epithelium , Mouse , D13A1 , 165Ho , 1:250 , Fluidigm , .

Techniques: Software, Generated

Journal: Frontiers in Physiology

Article Title: Using Imaging Mass Cytometry to Define Cell Identities and Interactions in Human Tissues

doi: 10.3389/fphys.2021.817181

Figure Lengend Snippet: List of antibodies used on human kidney tissue.

Article Snippet: β-Catenin , Tubular epithelium , Mouse , D13A1 , 165Ho , 1:250 , Fluidigm , .

Techniques: Membrane