Adult samples were fixed in 4% paraformaldehyde in 0.1 M Phosphate buffer overnight and decalcified with 0.5M EDTA for 3 days to 1 week at 4 °C. Seven μm paraffin sections were prepared. Sections were treated for antigen retrieval with 0.01M Citrate buffer (pH 6.0) by microwaving for 6 minutes. Immunohistochemistry was performed according to (Jiang et al., 1998 ) using the following antibodies (1: 200 dilution): β-catenin (Sigma; C2206, St. Louis, MO); PCNA (Chemicon; CBL407, Billerica, MA); NCAM and tenascin-C (Chuong and Chen, 1991 (link)). Secondary antibodies were either biotinylated anti mouse IgG or anti rabbit IgG (Vector Laboratories, 1:200 dilution). The tertiary antibody was streptavidin (Vector Laboratories, 1:200 dilution). An AEC substrate kit (Vector Laboratories) was used to develop the staining. Hematoxylin was used to perform faint counterstaining. BrdU staining was performed according to (Wu et al., 2004 (link)). For BrdU (BD; 347580; 1:200 dilution) / β-catenin or PCNA/β-catenin double staining, secondary antibodies, Alexa Fluor anti-Rabbit-488 (A11008) and anti-mouse-546 (A11030) from Invitrogen (Grand Island, NY) were used at a 1:200 dilution. DAPI was used to visualize the nuclei. Stained sections were imaged with a Zeiss 510 confocal microscope.
β catenin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
β-catenin is a protein that plays a crucial role in cell-cell adhesion and signal transduction. It is a key component of the Wnt signaling pathway, which is involved in various cellular processes, including cell proliferation, differentiation, and migration. β-catenin is widely used in research applications to study these fundamental biological mechanisms.
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Lab products found in correlation
E cadherin, by Thermo Fisher Scientific (11 mentions)
N cadherin, by Thermo Fisher Scientific (5 mentions)
β catenin, by Cell Signaling Technology (4 mentions)
E cadherin, by Cell Signaling Technology (3 mentions)
Gapdh, by Thermo Fisher Scientific (3 mentions)
β actin, by Thermo Fisher Scientific (3 mentions)
Ab205719, by Abcam (3 mentions)
Vimentin, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (2 mentions)
Proliferating cell nuclear antigen (pcna), by Thermo Fisher Scientific (2 mentions)
Proliferating cell nuclear antigen (pcna), by Merck Group (2 mentions)
β catenin, by Merck Group (2 mentions)
Alexa fluor anti rabbit 488 a11008, by Thermo Fisher Scientific (2 mentions)
Anti mouse 546 a11030, by Thermo Fisher Scientific (2 mentions)
Aec substrate kit, by Vector Laboratories (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
58 protocols using β catenin
1
Immunohistochemical Analysis of Developmental Markers
2
Immunohistochemistry Characterization of Tissue Samples
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Adult samples were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer overnight and decalcified with 0.5 mol/L ethylenediaminetetraacetic acid for 3 days to 1 week at 4°C. Seven micron paraffin sections were prepared. Sections were treated for antigen retrieval with 0.01 mol/L citrate buffer (pH 6.0) by microwaving for 6 min. Immunohistochemistry was performed according to Jiang et al. (1998 ) using the following antibodies (1:200 dilution): β‐catenin (Sigma; C2206, St Louis, MO); PCNA (Chemicon; CBL407, Billerica, MA); NCAM and tenascin‐C (Chuong and Chen 1991 ). Secondary antibodies were either biotinylated anti‐mouse IgG or anti‐rabbit IgG (Vector Laboratories, Burlingame, CA, USA; 1:200 dilution). The tertiary antibody was streptavidin (Vector Laboratories, 1:200 dilution). An AEC substrate kit (Vector Laboratories) was used to develop the staining. Hematoxylin was used to perform faint counterstaining. BrdU staining was performed according to Wu et al. (2004 ). For BrdU (BD; 347580; 1:200 dilution)/β‐catenin or PCNA/β‐catenin double staining, secondary antibodies Alexa Fluor anti‐rabbit‐488 (A11008) and anti‐mouse‐546 (A11030) from Invitrogen (Grand Island, NY) were used at a 1:200 dilution. 4’,6‐Diamidino‐2‐phenylindole (DAPI) was used to visualize the nuclei. Stained sections were imaged with a Zeiss 510 confocal microscope.
3
Quantifying Gene Expression in Liver Tissue
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From liver biopsy specimens, a 3-mm-long sample was saved from each subject to measure gene expression [23 (link)]. Total RNA was isolated from the samples using a ReliaPrep™ RNA Cell Miniprep System (Promega, Madison, WI, USA). Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) to produce cDNA, according to the manufacturer’s instructions. Real-time PCR was performed using a StepOnePlus™ Sequence Detection System (Applied Biosystems) with the TaqMan Universal PCR Master Mix reagent (Applied Biosystems).
From liver tissue, we determined mRNA levels of IRS1, IRS2, β-catenin, GCK, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Target values were normalized to the expression of GAPDH.
Primers and probes for IRS1 (Hs.471508), IRS2 (Hs.442344), β-catenin (Hs.476018), and GCK (Hs.1270) were purchased from an assay-on-demand facility (Applied Biosystems) as follows: IRS1, Hs00178563_m1; IRS2, Hs00275843_s1; β-catenin, Hs00355049_m1; GCK, Hs01564555_m1; GAPDH, 4326317E (Applied Biosystems).
From liver tissue, we determined mRNA levels of IRS1, IRS2, β-catenin, GCK, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Target values were normalized to the expression of GAPDH.
Primers and probes for IRS1 (Hs.471508), IRS2 (Hs.442344), β-catenin (Hs.476018), and GCK (Hs.1270) were purchased from an assay-on-demand facility (Applied Biosystems) as follows: IRS1, Hs00178563_m1; IRS2, Hs00275843_s1; β-catenin, Hs00355049_m1; GCK, Hs01564555_m1; GAPDH, 4326317E (Applied Biosystems).
4
Evaluation of β-catenin Interactome
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For β-catenin-CBP/P300/E-cadherin/N-cadherin coimmunoprecipitations, MGC-803 cells were incubated 48 h with or without ICG-001 and 100 μg protein extract was diluted to 1 mL in coimmunoprecipitation (Co-IP) buffer (Thermo, USA). 2 μg of β-catenin (Santa Cruz Biotechnology, USA) antibody was added to the protein samples and the mix were incubated overnight at 4 °C with rotation. 20 μL of 50% protein A/G-agarose bead slurry (equilibrated in Co-IP buffer) were added, and after 2 h–incubation at 4 °C, the beads were washed 4 times with Co-IP buffer (1 ml per wash) and diluted with 1× loading buffer. Western blotting was performed, and CBP (Santa Cruz Biotechnology, USA), P300 (Santa Cruz Biotechnology, USA), E-cadherin (Cell Signaling Technology, USA), N-cadherin (Cell Signaling Technology, USA) were detected.
5
Immunofluorescence Staining of Cell Markers
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Cells were grown to confluency and fixed with 4% paraformaldehyde for 10 min at room temperature, blocked with 1% BSA, and then incubated with one of the primary antibodies including LEF1, PD-L1, β-catenin, VE-cad, Vim or Ki67 (ThermoFisher Scientific) at 4 °C overnight. After washing with PBS, the cells were incubated with an Alexa 488 or a Cy3 secondary antibody at room temperature for 1 h. Images were detected by a confocal laser scanning microscope (Leica).
6
siRNA Knockdown Optimization Protocol
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siRNA for E‐cadherin (#4392420), β‐catenin, and the Silencer Select Negative Control #1 siRNA (Scramble, #4390843) were purchased from Thermo Scientific as SMARTpools. YAP1 silencer® select siRNA was also purchased from Thermo Scientific (ID: s20368). For siRNA transfections, cells were transfected with these oligos using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After transfection, efficiency was determined through western blot or RT‐qPCR.
7
Generating Ctnnb1 Knockout Cells
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RNA sequences targeting exon 4 of mouse catenin (cadherin associated protein), beta 1 (Ctnnb1) were designed using the web-based CHOPCHOP platform55 (link): Ctnnb1_gRNA1: 5′- GATTAACTATCAGGATGACG-3′. The gRNA sequence was inserted into the pSPCas9(BB)-2A-GFP vector (PX458, 48138, Addgene56 (link)) for transfection into tumor cells using polyethylenimine (408727, Sigma Aldrich). After 24 h, GFP-expressing cells were single-cell sorted (BD FACSAria II, BD Biosciences). Genomic DNA was isolated from individual clones, the relevant gene fragment was amplified by PCR (Forward primer: 5′-GTTCCCTGAGACGCTAGATGAG-3′. Reverse primer: 5′-ACATCACTGCTTACCTGGTCCT-3′.) and screened by Sanger sequencing for non-sense mutations in Ctnnb1. Additional verification of gene knockout was performed by immunofluorescence staining (primary antibody β-catenin [610153, 1:500, BD Biosciences] and secondary antibody goat anti-mouse IgG Alexa 488 [A28175, 1:500, Thermo Fisher Scientific]) and western blot analysis (β-catenin, 1:500).
8
Chromatin Immunoprecipitation and RNA Sequencing
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ChIP-seq were performed as described in previously [4 (link),6 (link)]. Briefly, cells were dual cross-linked with 2 mM ethylene glycol bis (succinimidyl succinate) (EGS) and disuccinimidyl glutarate (DSG) and 1% formaldehyde. Chromatin was isolated and then fragmented with 60 units MNase (New England Biolabs, M0247S) at 37 0C for 10 minutes. For β-catenin ChIP-seq, cells were first fractionated by Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher, Cat# 78840) to obtain a nuclear fraction that was diluted 1:5 with MNase digestion buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1% Triton X-100, 1 mM CaCl2, 1 mM DTT), and then digested with MNase as above. Antibodies used in the ChIP-seq experiments were against β-catenin (Cell Signaling, Cat#9581), WDR77 (SCBT, Cat# sc-100899), and MafB (SCBT, Cat# sc-10022).
For RNA-seq experiments, mRNeasy Mini Kit (Qiagen, No. 217004) was used for RNA extraction. 0.4 μg total RNA was used for RNA-seq library construction by a TruSeq RNA Library Prep Kit V2 (Illumina, RS-122-2001). Both ChIP-seq and RNA-seq libraries were sequenced at the Bauer Core Facility, Harvard.
For RNA-seq experiments, mRNeasy Mini Kit (Qiagen, No. 217004) was used for RNA extraction. 0.4 μg total RNA was used for RNA-seq library construction by a TruSeq RNA Library Prep Kit V2 (Illumina, RS-122-2001). Both ChIP-seq and RNA-seq libraries were sequenced at the Bauer Core Facility, Harvard.
9
Modulating SOX17 and β-Catenin Signaling
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The following antibodies were used: c-Myc (9E10) (sc-40, Santa Cruz Biotechnology, Santa Cruz, CA); SOX17 (AF1974, R&D systems, Minneapolis, MN); pan-cytokeratin (sc-81714, Santa Cruz Biotechnology, Santa Cruz, CA); β-catenin (zymed 13-8400, Thermo Fisher, Waltham, MA) vinculin (v4139, Sigma-Aldrich, St. Louis, MO); HRP-anti-mouse IgG (NA934V, GE Healthcare); Alexa Fluor-488 goat anti-mouse IgG (H+L) (A11029, Invitrogen, Carlsbad, CA), Alexa Fluor-488 donkey anti-goat IgG (H+L) (705-546-147, Jackson ImmunoResearch Laboratories, West Grove, PA); Alexa Fluor-594 donkey anti-mouse IgG (H+L) (715-586-150, Jackson ImmunoResearch Laboratories, West Grove, PA).
The coding sequencing of WT SOX17 was PCR-amplified from human tumor cDNA synthesized using superscript III reverse transcriptase (Thermo Fisher, Waltham, MA), and cloned into the pCDH-CMV-MCS-EF1-GreenPuro plasmid (SystemsBiosciences Palo Alto, CA). Mutations were introduced using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). A mutant β-catenin (S33A, S37A, T41A, S45A) expression construct, SOX17 compressed motif reporter [16 (link)] and pBAR TCF/β-catenin activated reporter [35 (link)] were obtained from addgene.
The coding sequencing of WT SOX17 was PCR-amplified from human tumor cDNA synthesized using superscript III reverse transcriptase (Thermo Fisher, Waltham, MA), and cloned into the pCDH-CMV-MCS-EF1-GreenPuro plasmid (SystemsBiosciences Palo Alto, CA). Mutations were introduced using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). A mutant β-catenin (S33A, S37A, T41A, S45A) expression construct, SOX17 compressed motif reporter [16 (link)] and pBAR TCF/β-catenin activated reporter [35 (link)] were obtained from addgene.
10
Protein Expression Analysis in Tumor Samples
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Tumors were excised and homogenized in cold lysis buffer. The supernatants were collected from tissue homogenates and cell lysates for protein quantification. Protein (30 μg) was electrophoresed and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Subsequently, membranes were blocked and incubated with rabbit TAF15 (1:2000), PTBP1 (1:1000; Thermo Fisher Scientific), E-cadherin (1:1000), N-cadherin (1:1000), Slug (1:2000; Thermo Fisher Scientific), MMP-9 (1:1000), β-catenin (1:1000), c-Myc (1:5000), Cyclin D1 (1:1000), DKK1 (1:1000), LGR5 (1:1500), and GAPDH (1:5000; Thermo Fisher Scientific) antibodies. Primary antibodies were provided by Abcam unless otherwise indicated. Membranes were washed and incubated with horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G antibody. The ECL substrate (Bio-Rad) was added to visualize the bands, and band intensity was analyzed with the Image J software.
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