Wnt/β-catenin pathway inhibitors FH535, ICRT14, IWP-2, and niclosamide were purchased from Sigma-Aldrich, USAFH535, ICRT14, IWP-2, and niclosamide (10, 25, and 50μM each) were dissolved in the 0.5% dimethyl sulfoxide (DMSO). The data recorded from the migrations and proliferation with Wnt/β-catenin pathway inhibitors were compared against control group (DMSO 0.5%).
Icrt14
Manufactured by Merck Group
Sourced in United States
The ICRT14 is a laboratory instrument designed for ion chromatography analysis. It is capable of separating and analyzing ionic species in a variety of sample types. The ICRT14 provides reliable and accurate results, making it a versatile tool for researchers and analysts in various fields.
Automatically generated - may contain errors
Lab products found in correlation
Niclosamide, by Merck Group (3 mentions)
Icrt3, by Merck Group (3 mentions)
Anti β catenin, by BD (2 mentions)
Bafilomycin, by Merck Group (2 mentions)
Pyrvinium, by Merck Group (2 mentions)
Icg 001, by R&D Systems (2 mentions)
Xav939, by Cayman Chemical (2 mentions)
Pcdna5 to plasmids, by Merck Group (2 mentions)
Nsc668036, by Merck Group (2 mentions)
Cct031374, by R&D Systems (2 mentions)
Icrt5, by R&D Systems (2 mentions)
Lithium chloride (licl), by Merck Group (2 mentions)
Quercetin, by Merck Group (2 mentions)
Fh535, by Merck Group (1 mentions)
Iwp 2, by Merck Group (1 mentions)
Adipored assay reagent, by Lonza (1 mentions)
Ki 67, by Gene Tech (1 mentions)
Anti β actin, by ZSGB-BIO (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Pyrvinium pamoate, by Merck Group (1 mentions)
14 protocols using icrt14
1
Wnt/β-catenin Pathway Inhibitor Assay
2
Immortalized Adipocyte Differentiation Assay
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SVCs were isolated from WAT biopsies, cultured, and differentiated as described (Collins et al., 2010 (link)) (see Supplemental Information ). SVCs isolated from a male subject were immortalized by co-expressing human telomerase reverse transcriptase and human papillomavirus type-16 E7 oncoprotein (Pinnick et al., 2014 (link)). To study the effects of iCRT14 (Sigma-Aldrich) on adipogenesis, SVCs were seeded in type I collagen-coated 96-well plates. Culture media were replaced after 24 hr with growth media containing iCRT14 (1 μM or 10 μM) or DMSO. Confluent cells were differentiated 48 hr later in the same concentration of iCRT14 (or DMSO) throughout for 21 days. Media were changed every 2–3 days. Intracellular lipid levels were quantified using the AdipoRed assay reagent (Lonza) and a CytoFluor Multi-Well Plate Reader series 4000 (PerSeptive Biosystems).
3
Modulating Embryonic Development with BMP2 and GSK3β Inhibitors
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Embryos were treated with recombinant hBmp2 (a gift from Dr Wölfl) at concentrations from 0.1 to 1 μg ml−1. As a control, 1 μg ml−1 bovine serum albumin (BSA) was applied. hBmp2 protein was added at one-two-cell-stage embryos and cDNAs were prepared at the blastula (12–14 h.p.f.), gastrula (30 h.p.f.) or planula (74 h.p.f.) stages. For WISH experiments, hBmp-treated blastulae and gastrulae (12 h.p.f. (blastula) and 24 h.p.f. (gastrula) were fixed. For experiments using the GSK3β inhibitors to enhance β-catenin signalling, embryos were treated with ALP, BIO or 1-AZA (Sigma) at the concentrations indicated. cDNAs were prepared at the blastula and early gastrula stages (12–18 h.p.f.) for the qPCR analyses. For immunostaining of neuropeptides and mOrange protein, embryos were treated with GSK3β inhibitors from one- and two-cell stage to blastula stage. After being washed, the embryos were cultured in Nematostella medium until early (72 h.p.f.) or late (98 h.p.f.) planula stages. To pharmacologically inhibit β-catenin signalling, embryos were treated with 10 μM or 50 μM iCRT14 (Sigma), a Tcf/β-catenin inhibitor, for 14 h (blastula), 3 days (early planula) or 4 days (late planula).
4
Investigating P2Y2 and Wnt Signaling
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ATP and iCRT‐14 were purchased from Sigma (St Louis, MO, USA). ATP was dissolved in sterile normal saline and used at a concentration of 100 μM. The iCRT‐14 was dissolved in DMSO and used at a concentration of 25 μM. The antibodies used in this study are as follows: anti‐β actin (TA‐09, ZSGB‐BIO, Beijing, China), anti‐P2Y2 (H‐70; SC‐20124, Santa Cruz, CA, USA), anti‐β‐catenin (61053, BD Biosciences, San Jose, CA, USA), anti‐Myc (K422; BS2462, Bioword Technology, St Louis, MN, USA), anti‐Axin2 (E2A6978, Enogene, Nanjing, China), anti‐CD44 (ZM‐0537, ZSGB‐BIO, Beijing, China), Ki‐67 (GM001, Gene Tech, Shanghai, China).
5
Comprehensive Antibody Panel for Cancer Research
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Antibodies purchased from various sources as follows: β-actin-HRP, c-Myc and uPA, (Abcam); N-cadherin, SMAD2/3, p21WAF1, and p53 (Cell Signaling); β-catenin, Wnt1, E-cadherin, Survivin, Vimentin, Fibronectin, Kinesin, VEGF, MMP2, MMP9, Cyclin D1, hnRNP-K, TGF- β1, and rabbit-/mouse-HRP (Santa Cruz); and mouse/rabbit Alexa Fluor 488- and/or 568-conjugated secondary antibodies and Hoechst 33342 (Invitrogen) additional details provided in Supplementary Information (Table S1 ). Anti-CARF antibody (Clone FL-A10) was raised indigenously in the laboratory15 (link). CARF siRNA (Silencer® Select & In vivo Ready Oligos) were purchased from Thermo Fisher. Wnt and β-catenin inhibitors, i.e. IWP2 (Santa Cruz), Pyrvinium pamoate (PP) and iCRT14 (Sigma-Aldrich) were purchased. Tissue microarray slides with embedded clinical tumor (various normal/cancer and breast cancer samples of histology, stage, age) were procured from SuperBioChip (Tissue-Array, South Korea) Laboratories.
6
Canonical Wnt Signaling Modulation
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Short hairpin RNAs (shRNA), cDNAs encoding human WNT7A and FGF1, and pcDNA5/FRT/V5-His, pcDNA6/TR and pcDNA5/TO plasmids were purchased from Sigma-Aldrich, Thermo Scientific (Rockford, IL, USA) and Life Technologies (Life Technologies, Grand Island, NY, USA), respectively. Niclosamide, iCRT3, iCRT14, Pyrvinium, Bafilomycin, Quercetin, NSC668036 and LiCl were purchased from Sigma-Aldrich. XAV939 and IWR were purchased from Cayman Chemical (Ann Arbor, MI, USA). IWP and Box5 were purchased from Thermo Scientific. ICG001, CCT031374 and iCRT5 were obtained from R&D Systems (Minneapolis, MN USA).
7
BVDV Inhibition in Bovine Cells
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BPC were plated in 24 wells for 3 days at 37 °C at a starting density of 3 × 105 cells/well in triplicate. Cells were inoculated at a MOI of 1 with BVDV-1 NADL and treated with 25 µM of iCRT14 (Sigma-SML0203) or 10 µM FZM1 (Merck-534358) at 1-, 2- or 3-h post-infection. The supernatants were harvested at 24 h post-infection and titred, in FAID50/mL, on MDBK with a pool of anti-Pestivirus monoclonal antibodies (BI collection) and Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 488 (Invitrogen).
The CPE were observed on BPC under microscope in white light.
The CPE were observed on BPC under microscope in white light.
8
Modulating Wnt Signaling in Embryos
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To activate/inhibit cWnt signaling, gastrulating embryos were treated with 0.5/1/2.5 μM 1-Azakenpaullone (Sigma, Canada/China) or 1/2.5/10 μM iCRT-14 (Sigma, USA/China) respectively. Stock solutions were prepared with DMSO at 10 mM, aliquoted and stored at − 20 °C. Working solutions were prepared before use by dilution of stock solutions in filtered seawater (FSW) to the final concentration. Control embryos were exposed to 0.1% DMSO in FSW. Working solutions were refreshed daily. Incubation was performed in the dark.
9
Assessing iCRT14 Cytotoxicity in Kidney and Skin Cells
10
RSV Infection Modulation by Wnt and HBD3
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Cells were infected with RSV and mKate2-RSV at the multiplicity of infection (MOI) of 1 or 3. Virus adsorption was performed for 1.5 h (at 37°C) in OPTI-MEM medium (Gibco). Following adsorption, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS; Gibco) and infection was continued in the presence or absence of a complete medium. In some experiments, cells were pre-treated with either vehicle (DMSO) or Wnt/β-catenin pathway inhibitors (iCRT3 or iCRT14; 25uM; Sigma) and infected with RSV in the absence and presence of these inhibitors. For human beta-defensins 3 (HBD3) treatment studies, A549 cells were treated with 10ug/ml of purified HBD3 (Peprotech) or vehicle (PBS with 0.1% BSA). Cells were also treated with either lithium chloride (LiCl; Sigma) or vehicle (water) for 24 h. A549 cells were transfected with either empty-FLAG plasmid or FLAG-tagged LRP5 (FLAG-LRP5; 200 ng/ml) by using Lipofectamine 2000 (Life Technologies).
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