Colorectal cancer cell lines and primary colorectal cancer cells were seeded into 24-well low-adhesion plates (Corning, NY, USA) at 200 cells/well and cultured in serum-free sphere medium (containing 1× B27, 20 μg/L EGF, 20 μg/L bFGF, 4 mg/L insulin, 0.4% BSA, and 200 IU/mL streptomycin). These cells were grown in the presence or absence of Wnt Pathway Inhibitor XI, Wnt/β-catenin Inhibitor, Cardamonin (Merck, Germany), or the Wnt activator recombinant Wnt3a (R&D Systems, MN, USA) for 7–14 days at 37 °C in a humidified atmosphere containing 5% CO2. After the incubation period, the spheres were dilute passaged for an additional 1 week and the number of spheres was counted manually.
Recombinant wnt3a
Manufactured by R&D Systems
Sourced in United States, United Kingdom
Recombinant Wnt3a is a laboratory reagent produced by R&D Systems. It is a secreted glycoprotein that functions as a signaling molecule involved in various cellular processes. The product is provided as a purified recombinant protein.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by Cell Signaling Technology (4 mentions)
Dual luciferase reporter assay system, by Promega (4 mentions)
Wnt5a, by R&D Systems (4 mentions)
Wnt3a, by R&D Systems (4 mentions)
Neurobasal growth medium, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Ankirin g, by Cell Signaling Technology (3 mentions)
Map1b, by Cell Signaling Technology (3 mentions)
Lithium chloride (licl), by Merck Group (3 mentions)
Wnt7a, by R&D Systems (3 mentions)
Gsk3β ser9, by Cell Signaling Technology (2 mentions)
Cytation 5 multi mode reader, by Agilent Technologies (2 mentions)
Hek293 tcf lef reporter recombinant cell line, by BPS Biosciences (2 mentions)
One step luciferase assay system, by BPS Biosciences (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Alexa 647 conjugated anti β catenin antibody l54e2, by Cell Signaling Technology (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
56 protocols using recombinant wnt3a
1
Colorectal Cancer Sphere Assay
2
Wnt Pathway Modulation in Neurons
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Antibodies against ATF2, MAP1B, Tau, Ankirin G, β-catenin, GSK-3 β(ser9), β-actin, MAP2 and phalloidin were obtained from Cell Signaling Technology, Inc. (Trask Lane, Danvers, MA). Neurobasal growth medium was obtained from Life Technologies (Carlsbad, CA). Other reagents were purchased from Sigma (St. Louis, MO). Wnt-C59 inhibitor was dissolved in DMSO and added to neuronal cultures at a final concentration of 0.01% DMSO or less (Cellagen Technology, San Diego, CA). Recombinant Wnt3a, Wnt5a, and Wnt7a (R&D Systems, Abingdon, UK) were used at 50 ng/ml, 100 ng/ml, and 200 ng/ml, respectively with corresponding dilutions in vehicle.
3
Wnt Signaling Activation in HEK293 Cells
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The HEK293 TCF/LEF-reporter recombinant cell line (BPS Bioscience; Cat.# 60,501) was cultured according to the vendor’s recommendations. To avoid detachment of the reporter cells from the culture well surface, L-WRN CM or primary culture medium was carefully added at 1:1 vol/vol to the existing HEK293 medium in the culture well for this assay. Viability in the 1:1 medium mixtures was determined using the CellTiter-Glo® 3D Cell Viability Assay, as described above. Recombinant Wnt3a (R&D systems; Cat.# P27467) was used as a positive control. L-WRN CM batches were tested at a final concentration of 5% (i.e., added 10% L-WRN CM at a 1:1 vol/vol to the HEK293 medium) for Wnt activity assays. Luciferase activity was determined using the ONE-Step™ Luciferase Assay System (BPS Bioscience; Cat.# 60690–2) and luminescence was measured with a Cytation5 Multi-mode Reader (BioTek). Culture wells containing L-WRN CM and ONE-Step™ reagent but without cells were used to determine the average back-ground luminescence. Culture wells containing cells, and HEK293:primary culture medium (1:1) were used to determine baseline reporter activity. Experiments included 3 technical replicates, and 3 biological experiments with distinct passages of cells were performed. Technical replicates from each experiment were averaged prior to statistical analysis.
4
Wnt3a-Mediated Signaling Pathway
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Recombinant Wnt3a (R&D Systems), LiCl (Sigma-Aldrich), oxaliplatin (Oxal; Sigma-Aldrich), 5-fluorouracil (Sigma-Aldrich), and cycloheximide (Sigma-Aldrich) were commercially obtained. PLD1 inhibitor was purchased from Cayman Chemical. Dual-luciferase assay kits were purchased from Promega.
5
Wnt3a Transwell Migration Assay
6
Cell Culture of Lung Cancer and Control Cell Lines
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Lung cancer cell lines (A549 and PC9), HEK293T cell line and normal bronchial epithelial cell line (BEAS2B) were purchased from cell banks of Shanghai Institutes of Biological Sciences (Shanghai, China). A549, HEK293T and BEAS2B cell lines were cultured in DMEM (HyClone, Logan, Utah, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). PC9 cells were cultured in RPMI 1640 medium (HyClone) with 10% FBS and antibiotics. All the cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Recombinant Wnt3a was purchased from R&D Systems (Minneapolis, MN, USA).
7
Quantifying β-catenin Accumulation in K562 Cells
8
CRISPR-Mediated Generation of Evi-Deficient HCT116 Cells
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HCT116Evi_KO cells were generated using CRISPR/Cas9 following a protocol described in reference 44 (link). In short, the HCT116 cells were transfected with px459 vector (a gift from Feng Zhang, Addgene plasmid # 48139; http://n2t.net/addgene:48139 ; RRID: Addgene_48139) expressing Cas9 and sgRNA targeting human Evi/Wls (5′-GTAAGCCAGGGAAACGTCCA-3′). After 48 h, the cells were selected with 2 μg/ml of puromycin (Invitrogen) for 72 h. Because Evi silencing in HCT116 cells resulted in growth inhibition (16 (link)), either recombinant Wnt3a (50 ng/ml; R&D Systems) and/or medium from parental cell lines was added to the growth medium. Single-cell clones were selected and were used for further analysis. The generation of HCT116Evi_KO cells will be described in detail in Voloshanenko et al (manuscript in preparation).
9
Lung Cancer Cell Demethylation with 5-aza-dC
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Cultivated lung cancer cells were treated with different concentrations of 5-aza-2′-deoxycytidine (5-aza-dC) (Sigma) dissolved in culture medium every 24 hours. Using q-MSP and MTT assays, the drug concentration required for β-catenin promoter CpG island demethylation with no significant effect on cell growth was selected (7 µM) and cells were collected after continued culture for 48 hours (Figure S1 ).
Cells were treated with 500 ng/ml recombinant Wnt3a (R&D Systems, Minneapolis, MN, USA) for 48 hours.
Cells were treated with 500 ng/ml recombinant Wnt3a (R&D Systems, Minneapolis, MN, USA) for 48 hours.
10
Real-time Neuroblastoma Cell Proliferation
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To monitor proliferation in real time, neuroblastoma cells were seeded in a 96-well plate, in quadruplicates, in media containing 1% (v/v) FBS (5000 cells/well). After incubation for 24 hours, cells were treated with recombinant Wnt3a and/or R-Spondin2 (R&D Systems, 100 ng/mL each) using low serum conditions and transferred to the IncuCyte ZOOM live cell imaging system (Essen BioScience). Epidermal growth factor (Sigma) was used at 10 ng/mL. Images were taken in four different fields in each well, every two hours and phase confluence was calculated as a surrogate for growth at each time point. To measure cell death, DRAQ7 (BD Biosciences), a dye that is taken up by dead cells only and fluoresces at 678 nm, was added at 1.5 μM concentration and fluorescence was measured in the red channel. To measure migration, cells were seeded at near confluent levels (50000 cells/well) in ImageLock 96-well plates (Essen BioScience), in quadruplicates, in media containing 1 % FBS (v/v). Cells were stimulated with Wnt3a and/or R-Spondin2 24 hours later, and, following another 24 hours, scratched by using the WoundMaker tool (Essen BioScience) according to manufacturer’s instructions. Migration was monitored at every 2 hours automatically, and wound width and relative wound density were calculated for every time point.
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