Journal: Biomedicines

Article Title: Bacteroides fragilis Toxin Induces Intestinal Epithelial Cell Secretion of Interleukin-8 by the E-Cadherin/β-Catenin/NF-κB Dependent Pathway

doi: 10.3390/biomedicines10040827

Figure Lengend Snippet: β-catenin gene disruption by CRISPR-Cas9 in HT29/C1 cells. ( A ) Validation of CTNNB1 knockout in HT29/C1 cells. Genomic DNA of wild type (WT) and β-catenin knockout HT29/C1 cells (KO#1 and KO#2) were amplified by nested PCR and sequencing analysis of CTNNB1 was conducted. The CRISPR-Cas9 system introduced an indel mutation (red) in the target sites of the CTNNB1 . ( B ) Immunofluorescence imaging of WT, KO#1, and KO#2 cells. E-cadherin (green) and β-catenin (red). Nuclei were stained with DAPI (blue), and images were taken with a confocal laser-scanning microscope (×1640). ( C ) Western blot analysis of β-catenin in WT and β-catenin knockout HT29/C1 cells. GAPDH was used as an internal control. ( D ) Cellular proliferation of WT and β-catenin knockout HT29/C1 cells. Viable WT, KO#1 and KO#2 cells were enumerated by the trypan blue exclusion assay. * p < 0.05 vs. WT (unpaired t -test).

Article Snippet: CTNNB1 gRNA-lentiCRISPR v2 vector was co-transfected with pMD2.G (Addgene, Cambridge, MA, USA) and psPAX2 (Addgene, Cambridge, MA, USA) into HEK293T cells to produce lentiviruses containing CTNNB1 gRNA-lentiCRISPR v2.

Techniques: CRISPR, Knock-Out, Amplification, Nested PCR, Sequencing, Mutagenesis, Immunofluorescence, Imaging, Staining, Laser-Scanning Microscopy, Western Blot, Trypan Blue Exclusion Assay

Journal: Cancer Cell International

Article Title: Intestinal region-specific Wnt signalling profiles reveal interrelation between cell identity and oncogenic pathway activity in cancer development

doi: 10.1186/s12935-020-01661-6

Figure Lengend Snippet: Model for Wnt activation. a Organoids were grown from 3 locations of the wild type (wt) murine intestine, and after transduction with stable Ctnnb1 S (Cβ) a cystic morphology can be observed in all locations. b Immunoblot of CTNNB1 and loading control GAPDH in organoids grown without (wt) or with transduced Ctnnb1 S (Cβ). c Number of reads matching the wild type (wt.) hotspot region of Ctnnb1 and the mutated (mut.) hotspot region of Ctnnb1 : c.97T > G;109T > G;121A > G;133T > G . d Quantitative PCR of Ctnnb1 and important Wnt target genes, n = 3, *p < 0.05, **p < 0.005 (Student’s t-test), mean fold change in vertical number. All error bars represent S.E.M. e Expression of Wnt target genes as average (n = 3) scaled count, centred around the location average. Right columns show summarized average logarithmic fold change (log2FC) of Cβ/wt and mean read count across locations. Indicated gene symbols are significantly differentially expressed in the average of all locations (adjusted p-value < 0.05, Wald -Test). f Gene Set Enrichment Analysis of Wnt target genes in each location separately using GSEA preranked mode or ( g ) testing enrichment of Hallmark gene sets across all locations using EGSEA

Article Snippet: A stabilized (mutated to p.S33A;S37A;T41A;S45A) version of Ctnnb1 (GenBank ID: NM_001165902.1:c.[97T > G;109T > G;121A > G;13T > G]) was obtained in pLX304 as a gift from William Hahn (Addgene # 42561) and cloned into the vector pWPXL downstream the constitutively active promotor EF1α (Addgene #12,257) which was kindly provided by Didier Trono Finally, P2A-dsRed sequence was cloned downstream Ctnnb1 S gene.

Techniques: Activation Assay, Transduction, Western Blot, Real-time Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

doi: 10.1101/661777

Figure Lengend Snippet: a) Schematic representation of murine β-catenin (Ctnnb1) locus and the two loxP alleles used for β-catenin studies in mESCs. Black boxes represent exons, yellow boxes coding exons, dashed red lines indicates loxP sites, white boxes represent exons excised upon CRE-mediated recombination of loxP sites. b) Western blot of NLC1 and SR18 cell lines upon 72hrs 4’-Hydroxytamoxifen treatment (+4OHT) and respective untreated controls (CTR). SR18 untreated cell line is heterozygous for full-length β-catenin deletion. c) β-catenin immunofluorescence staining on fixed SR18 (Ctnnb1 fl/del ) parental cell line (left) or upon 72 hours 4’-Hydroxytamoxifen treatment (right panel). DAPI was used to counterstain nuclei. A primary antibody raised against the C-terminal portion of β-catenin was used. d) Schematic representation of short-hairpin targeted regions (red triangles, β1, β2 and β3) and qRT-PCR amplicons (blue lines, #1 and #2) along the Ctnnb1 Birchmeier allele. e) Western blot of β-catenin of mESCs harbouring the Birchmeier β-catenin allele after (Ctnnb1 del/del (left)) or before (Ctnnb1 fl/fl (right)) CRE-mediated recombination of the lox-P sites. Cells were transduced with a control short-hairpin (shCtr) or three different short hairpins against β-catenin mRNA (β1, β2 or β3). f) qRT-PCR on total mRNA extracts of Ctnnb1 fl/fl or Ctnnb1 del/del cells transduced with the short-hairpin constructs used in . Two different amplicons were amplified to monitor deleted region (Ctnnb1 #1) or 3’UTR region (Ctnnb1 #3). GAPDH was used as housekeeping control. Error bars represents standard deviation of technical triplicates.

Article Snippet: sgRNAs were cloned by annealed oligos cloning into px459-SpCas9-Puro (Addgene # 48138) as previously described [ ], a list of the oligos used for generating Ctnnb1 targeting vectors is listed in . pSpCas9(BB)-2A-GFP (PX458) was a gift from Dr. Feng Zhang.

Techniques: Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, Transduction, Construct, Amplification, Standard Deviation

Journal: bioRxiv

Article Title: Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

doi: 10.1101/661777

Figure Lengend Snippet: a) SR18 Ctnnb1fl/del or Ctnnb1 del/del display overall similar alkaline phosphatase staining expression and morphology. BF=brightfield (scalebar=500 μm), PH=phase contrast (scalebar=100 μm). b) Immunofluorescence of Nanog and Oct4 on fixed SR18 Ctnnb1 fl/del or Ctnnb1 del/del cells. Scalebar=100 μM. DAPI was used to counterstain nuclei. c) Immunofluorescence of Plakoglobin on fixed SR18 Ctnnb1 fl/del or Ctnnb1 del/del cells. Scalebar=30 μM. DAPI was used to counterstain nuclei. d) Western blot of total protein extracts of SR18 Ctnnb1fl/del (SR18 ctr) or Ctnnb1 del/del (SR18 4OHT) cells. Protein extracts were probed for β-catenin (CTNNB1), Plakoglobin, Sox2, Oct4 and Nanog. Tubulin was used as loading control. e) Schematic representation of sgRNAs target positions along the β-catenin locus. sgRNAs were used in pairwise combinations to excise different gene regions. sgRNAs are represented as red arrows, indicating the position and orientation of oligonucleotides used for PCR genotyping (3 oligos PCR). f) PCR-genotyping of E14 mESCs transiently transfected with Cas9 and pairwise combinations of sgRNAs as depicted in e). Untransfected cells were used as parental control. Expected amplicon size is 824 bp for wild-type, 595 bp for sgRNA2+sgRNA3, 278 bp for sgRNA1+sgRNA3. g) Western blot for β-catenin on total protein extract of E14 mESCs parental cell line, or upon transient transfection of Cas9 and pairwise combination of sgRNAs as in e). Tubulin was used as a loading control. h) Quantification of full-lenght β-catenin deletion in g). β-catenin band intensity was normalized on Tubulin intensity for each sample and then rescaled as a percentage of the untrasfected parental cell line.

Article Snippet: sgRNAs were cloned by annealed oligos cloning into px459-SpCas9-Puro (Addgene # 48138) as previously described [ ], a list of the oligos used for generating Ctnnb1 targeting vectors is listed in . pSpCas9(BB)-2A-GFP (PX458) was a gift from Dr. Feng Zhang.

Techniques: Staining, Expressing, Immunofluorescence, Western Blot, Transfection, Amplification

Journal: bioRxiv

Article Title: Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

doi: 10.1101/661777

Figure Lengend Snippet: a) PCR genotyping of E14 transiently transfected with Cas9, sgRNA4 and sgRNA5 (right) and parental cell line (left). Three different oligos combinations were used to detect wild-type allele (F1+F2), deleted alleles (F1+R2) or both (F1+R1+R2). b) Sanger sequencing of Eβ11, Eβ15 and Eβ47 Ctnnb1 edited locus. Matching bases are represented as green letters, green dots are deleted bases. Orange arrows indicate expected Cas9 editing sites, sgRNAs sequences and PAM are shown as red arrows. c) Western blot of total protein extracts from E14, Eβ11, Eβ15 and Eβ47 mESCs. Protein extracts were probed for β-catenin, Plakoglobin, Nanog and Oct4 expression. Tubulin was used as loading control. d) Band intensity quantification relative to western-blot in figure (c). Band intensities were normalized on Tubulin intensity for each sample and then rescaled as fold-change with respect to the parental cell line. e) Flow cytometry analysis of E-Cadherin expression in Eβ11, Eβ15, Eβ47 and parental E14 cells. E14 cells undergoing neuro-ectodermal differentiation were used as negative control for E-Cadherin expression (top). f) Flow cytometry cell-cycle analysis on fixed E14, Eβ11, Eβ15 and Eβ47 cells. DAPI was used to measure DNA content. Data are represented as histogram depicting the percentage of cells in G1 (black), S (grey) or G2/M (light grey). One exemplificative experiment. g, h) Growth curve of E14, Eβ11, Eβ15 and Eβ47 cells and doubling time analysis. i) AP staining of E14, Eβ11, Eβ15 and Eβ47 cells cultured in Serum/LIF in presence of Vehicle (0.3 % DMSO) or 3 μM Chiron for 5 days. Whole plate scanning and magnification inset (dashed boxes). j) AP staining intensity quantification relative to . k) Immunofluorescence of parental E14 cells, Eβ11, Eβ15 and Eβ47 for Tcf3 expression. DAPI was used to counterstain nuclei. Scalebar= 50 μm.

Article Snippet: sgRNAs were cloned by annealed oligos cloning into px459-SpCas9-Puro (Addgene # 48138) as previously described [ ], a list of the oligos used for generating Ctnnb1 targeting vectors is listed in . pSpCas9(BB)-2A-GFP (PX458) was a gift from Dr. Feng Zhang.

Techniques: Transfection, Sequencing, Western Blot, Expressing, Flow Cytometry, Negative Control, Cell Cycle Assay, Staining, Cell Culture, Immunofluorescence

Journal: bioRxiv

Article Title: Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

doi: 10.1101/661777

Figure Lengend Snippet: a) Box plot of Ctnnb1 mRNA expression levels (raw counts) across WTV, WTC, KOV and KOC samples. b) Number of differentially expressed genes in various comparison relative to . c) Radar plot showing the fold-change of pluripotency and lineage marker genes in WTC (top panel, blue line) or KOV, KOC samples (light and dark red lines respectively, bottom panel), versus WTV sample (black line, top and bottom panel). d, e) Gene ontology analysis of biological processes (d) and KEGG pathways (e) enriched in differentially expressed genes in the KOV/KOC comparison (adjusted p.Value <0.05, absolute logFC >0.5) f) Histogram of counts per million (CPMs) of canonical Wnt target genes with minor expression level changes in KOV/KOC comparison. Individual biological replicates are shown.

Article Snippet: sgRNAs were cloned by annealed oligos cloning into px459-SpCas9-Puro (Addgene # 48138) as previously described [ ], a list of the oligos used for generating Ctnnb1 targeting vectors is listed in . pSpCas9(BB)-2A-GFP (PX458) was a gift from Dr. Feng Zhang.

Techniques: Expressing, Marker