Journal: bioRxiv

Article Title: Chromatin interaction maps identify Wnt responsive cis -regulatory elements coordinating Paupar-Pax6 expression in neuronal cells

doi: 10.1101/2021.05.18.442939

Figure Lengend Snippet: (A) N2A cells were transfected with pCl β-catenin S33Y or pCl empty vector and harvested for expression analysis 2 days later. Results are presented relative to the pCl control. (B, C) N2A cells were treated with either 50 ng/μl Wnt3a (B) or 0.1 ng/μl BMP4 (C) and harvested for RT-qPCR analysis 3 days later. 0.02% BSA in PBS was used as a negative control. (D) Genome browser graphic showing the ChIP amplified candidate CREs as well as the location of the TCF7L2 motifs and sgRNAs used in the CRISPRi experiment (GRCm38/mm10). (E) ChIP assays were performed in N2A cells using either an antibody against TCF7L2 or an isotype specific control. TCF7L2 occupancy at the indicated CREs was analysed by qPCR. Fold enrichment was calculated as 2 −ΔΔCt (IP/IgG) and is presented as mean value +/− sem. n≥3. One-tailed student’s t-test * p<0.05. (F) TCF7L2 levels were knocked-down in N2A cells by transfection of an endoribonuclease-prepared pool of TCF7L2 esiRNAs. An esiRNA pool targeting the luciferase gene was used as a negative control. Cells were harvested for expression analysis 3 days after transfection. (G) N2A cells were transfected with a plasmid co-expressing dCas9-KRAB and a sgRNA targeting the TCF7L2 motif within the indicated candidate CREs. A non-targeting sgRNA was used as a control. For all RT-qPCR reactions: Tcf7l2, Paupar and Pax6 expression levels were measured using RT-qPCR and results were normalised to Tbp. Results are presented as mean values +/− sem, n≥3. One-tailed student’s t-test * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: For Wnt and Bmp treatment, approximately 3 x 10 5 N2A cells were seeded per well in a 6-well plate in either growth medium containing 50 ng/ μl Wnt3a (R&D Biosystems, 5036-wn) or in low serum medium (DMEM supplemented with 5% FBS) containing 0.1 ng/ μl BMP4 (Thermo Fisher, PHC9534) as described in (Szemes, Melegh et al. 2020).

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Negative Control, Amplification, One-tailed Test, esiRNA, Luciferase

Journal: STAR Protocols

Article Title: Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier

doi: 10.1016/j.xpro.2021.100680

Figure Lengend Snippet: Preparation of Human monolayer EDMs media (100 mL) (stored at 4°C for 2–4 days)

Article Snippet: Embed the living stem cells into a basement membrane matrix (Matrigel) dome and cultured using 50% stem cell-enriched WRN conditioned media (WNT 3a, R-spondin, and Noggin) (prepared from L-WRN cells, ATCC CRL-3276 TM ) containing specific components such as Y27632 (ROCK inhibitor) and SB431542 (an inhibitor for TGF-β type I receptor), nicotinamide, A83-01 (an ACTIVIN /NODAL/TGF-β pathway inhibitor), Epidermal growth factor, and SB202190 (a p38 MAPK inhibitor) to support the growth and long-term cultures of 3D organoids ( , , , ).

Techniques: Concentration Assay

Journal: Oncotarget

Article Title: De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas

doi: 10.18632/oncotarget.9346

Figure Lengend Snippet: A . and B . Incubation of progenitor HepaRG cells with control or Wnt3a-conditioned medium for 13 days. A . Controls are hepatocyte-like. Cells incubated with Wnt3a are fibroblast-like. B . Coimmunodetection of the indicated proteins. Alpha smooth muscle actin (α-SMA), collagen IV and N-cadherin: green (FITC); albumin, red (FluoProbes 594); nuclei, blue (DAPI). Images were acquired with a Cellomics station at 20X. Wnt3a-incubated cells show bridging fascicules of spindle α-SMA(+)/albumin(−) cells. Stress fibers typical of myofibroblasts (white arrows) embrace foci of transitional elongated α-SMA(+)/albumin(+) cells (white arrowheads) , with granular and filamentous compartments (yellow arrowheads) . C . Gene expression kinetics of HepaRG cells incubated with recombinant Wnt3a or control BSA and analyzed by real-time PCR. D . FZD7_CRD and FZD8_CRD inhibit Wnt3a-induced Matrigel invasion. Cells were incubated in Matrigel-coated Boyden chambers with FCS(−) medium plus BSA; 7 nM Wnt3a; Wnt3a plus 30 nM FZD7 or FZD8_CRD. E . HepaRG cells invading Matrigel. F . Wnt3a enhances cell proliferation. Cells were incubated in 96-well plates with FCS(−) medium plus BSA or Wnt3a. BrdU(+) nuclei were counted with a Cellomics station.

Article Snippet: The lower chamber was filled with 800 μl of serum-free medium with or without recombinant mouse Wnt3a (R&D Systems).

Techniques: Incubation, Expressing, Recombinant, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas

doi: 10.18632/oncotarget.9346

Figure Lengend Snippet: A . Gene networks applying Ingenuity pathway analysis on the 358 gene set differentially regulated by Wnt3a. B . Wnt-induced differentiation of hepatic progenitors into fibrogenic myofibroblast-like cells matches the transcriptome signature of spontaneously emerging EMT in human HCC (GSE26391). 3P and 3SP human HCC cell lines derive from the same tumor, mesenchymal 3SP resulting from epithelial 3P cells that underwent spontaneous EMT in a human HCC . By gene set enrichment analysis, 3SP cells are significantly enriched in transcripts induced by Wnt3a in HepaRG cells. C . and D . Hierarchical clustering: 3SP and 3P cells are distinguished by C Wnt target genes from the Wnt Homepage and D Extracellular Wnt signaling (Wnt pathway ligands and receptors). Raw gene expression data were normalized by the Robust Multi-array Average (RMA) log 2 algorithm and quantile filtered.

Article Snippet: The lower chamber was filled with 800 μl of serum-free medium with or without recombinant mouse Wnt3a (R&D Systems).

Techniques: Expressing

Journal: Oncotarget

Article Title: De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas

doi: 10.18632/oncotarget.9346

Figure Lengend Snippet: A . RNA levels of the indicated genes in progenitor HepaRG HCC cells treated with Wnt3a after transfection with two β-catenin targeting siRNAs. B . LGR5 mRNA expression in HepaRG and Huh-7 cells treated with BSA (control), the Hedgehog pathway inducer Purmorphamine, Wnt3a and the Hedgehog inhibitor KAAD-cyclopamine. Tomatidine is a cyclopamine structural analog which does not inhibit the Hedgehog pathway. C . Immunodetection of LGR5 in HepaRG cells after 13 days of incubation with Wnt3a or BSA. LGR5, red (FluoProbes 594); nuclei, blue (DAPI). D . RNA expression of the hepatocyte differentiation marker ALDOB in HepaRG cells transfected with LGR5 siRNA. E . Gene networks generated by Weighted Correlation Network Analysis (WGCNA) using mRNA expression data for the indicated genes in 79 HCCs. Networks with a correlation coefficient threshold ≥0.30 plotted with Cytoscape . Closeness between nodes is proportional to the number of connections. Thickness of links is proportional to the correlation coefficients. F . 3D scatterplots showing the relationships between mRNA expression of the indicated genes in 79 HCCs. ACTA2, smooth muscle actin; COL4A1, procollagen, type IV, alpha 1 chain. Multiple regression coefficients (R) are indicated.

Article Snippet: The lower chamber was filled with 800 μl of serum-free medium with or without recombinant mouse Wnt3a (R&D Systems).

Techniques: Transfection, Expressing, Immunodetection, Incubation, RNA Expression, Marker, Generated

Journal: Oncotarget

Article Title: De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas

doi: 10.18632/oncotarget.9346

Figure Lengend Snippet: HepaRG progenitor cells were cultured with control FCS(−)/BSA(+) or FCS(−)/BSA(+)/Wnt3a. A . HAPLN1 mRNA levels 12 h and 13 days upon Wnt3a-induction. B . Immunodetection of HAPLN1, red (Cy5); N-cadherin, green (FITC). Nuclei, blue (DAPI). Images acquired at 20X. Control (BSA) shows very rare cells expressing HAPLN1 (arrow). HAPLN1 labels the pericellular matrix and colocalizes with N-cadherin.

Article Snippet: The lower chamber was filled with 800 μl of serum-free medium with or without recombinant mouse Wnt3a (R&D Systems).

Techniques: Cell Culture, Immunodetection, Expressing

Journal: PLoS Pathogens

Article Title: E6 proteins from high-risk HPV, low-risk HPV, and animal papillomaviruses activate the Wnt/β-catenin pathway through E6AP-dependent degradation of NHERF1

doi: 10.1371/journal.ppat.1007575

Figure Lengend Snippet: The listed E6 proteins were co-expressed with FLAG_E6AP_WT, the TOPFLASH or FOPFLASH luciferase reporter, and a renilla luciferase internal transfection control plasmid in C33A cells. Transfected cells were treated with Wnt3A conditioned media for 8.5 hours, lysed in 1X passive lysis buffer (Promega), and measured for luciferase and renilla luminescence. Fold activation was determined by normalizing the TOPFLASH luminescence by the FOPFLASH luminescence. Each E6 protein that could degrade NHERF1 (16E6_WT, 16E6_ΔPBM, 11E6_WT, and 18E6_WT) augmented the canonical Wnt pathway. 16E6_F69A/K72A, which cannot degrade NHERF1, failed to increase Wnt pathway activation over vector levels. Statistical significance was determined from three independent experiments by Student’s t-test (***<0.001, n.s. = no significance).

Article Snippet: 10% fetal bovine serum Wnt3A conditioned media was generated using L Wnt-3A murine fibroblasts (ATCC, CRL-2647) as previously described [ ].

Techniques: Luciferase, Transfection, Control, Plasmid Preparation, Lysis, Activation Assay