Serial dilutions of compounds were made in appropriate solvents (DMSO or H2O) and corresponding carrier controls were included in the assays. Serial dilutions were made for single soluble factor titrations; FGF4, sodium (ortho)vanadate (Sigma-Aldrich, S6508), PD0325901, PD98059 (Sigma, P215), activin A, TGFβ1, A83-01 (Tocris, 2939), Nodal (R&D systems, 1315-ND-025), SB431542 (Tocris, 1614), retinoic acid (Sigma-Aldrich, R2625), DL-epinephrine HCl (Sigma-Aldrich, E4642), 8Br-cAMP, SC144 (Tocris, 4963/10), IL6 (Peprotech, 216-16), IL11, Lif, BMP4 (Peprotech, 315-27), BMP7, LDN 193189 (Tocris, 6053), ML347 (Selleckchem, S7148), Noggin (Peprotech, 250-38), CHIR99021, IWP2 (Selleckchem, S7085) and XAV-939 (Selleckchem, S1180). Compounds with end concentrations that were used for XEn/Epi EpiC modulation: PI3K inhibitor ZSTK474 (Selleckchem, S1072) at 1 μM, insulin at 50 ng/ml, XAV-939 at 20 μM.
Xav939
Manufactured by Selleck Chemicals
Sourced in United States, China
XAV939 is a small molecule inhibitor that selectively targets the tankyrases, TNKS1 and TNKS2. It is a potent and selective inhibitor of tankyrase activity, which plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Selleck Chemicals (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (9 mentions)
Icg 001, by Selleck Chemicals (8 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (8 mentions)
Lithium chloride (licl), by Merck Group (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
S1180, by Selleck Chemicals (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Skl2001, by Selleck Chemicals (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Hct116, by American Type Culture Collection (3 mentions)
Sw480, by American Type Culture Collection (3 mentions)
Caco 2, by American Type Culture Collection (3 mentions)
Zstk474, by Selleck Chemicals (2 mentions)
134 protocols using xav939
1
Compound Serial Dilution Assays
2
Myocardial Ischemia/Reperfusion Injury Model
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Myocardial cell H/R treatment was established in H9c2 myocardial cells (from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) to simulate I/R injury model. For hypoxic incubation, H9c2 cells were cultured in glucose-free and serum-free DMEM medium and exposed to a humidified incubation chamber flushed with a gas mixture of 95% N2 and 5% CO2 at 37°C. After 6 hours of hypoxic incubation, the cells were transferred to high glucose DMEM medium containing 10% FCS (reoxygenation medium), and reoxygenated in 95% air and 5% CO2 at 37°C for 24 hours H9c2 cells were randomly divided into 5 groups1 (link): control group, cells cultured in 10% FBS-containing DMEM medium under normoxic conditions2 ; H/R group, cells subjected to H/R injury only3 (link); H/R+PBS group, cells subjected to H/R injury and 200 μL exosome-free PBS was added to the reoxygenation medium4 (link); H/R+ADMSCs-ex group, cells subjected to H/R injury and 200 μL PBS containing 400 μg of ADMSCs-ex were added to the reoxygenation medium; and5 (link) H/R+ADMSCs-ex+XAV939 group, cells subjected to H/R injury and 200 μL PBS containing 400 μg of ADMSCs-ex and Wnt/β-catenin inhibitors XAV939 (10 μM; Selleck, Houston, TX) were added to the reoxygenation medium.
3
Modulating Wnt/β-catenin Signaling
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U2-OS Cells were inoculated in a 96-well (5×103 cells/well) and were incubated for 24 h, and then transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, MA, USA), and 20 ng pRL-TK vector expressing Renilla luciferase (Promega, WI USA) following the recommended protocol using Lipofectamine 3000 (Thermo Scientific, MA, USA) for another 24 h. Subsequently, the assays were divided into four groups: resveratrol (0, 6 or 12 μg/ml resveratrol for 24 h), CHIR-99021 (inhibitor of GSK-3α/β, Selleck, TX, USA) + resveratrol (the cells were pretreated with 10 μm CHIR-99021 for 24 h to activate the Wnt/β-catenin signaling pathway); lentivirus infection (shCx43, NTC and Blank), and lentivirus infection + XAV939 [(inhibitor of β-catenin, Selleck, TX, USA), the cells were treated with 10 μm XAV939 for 24 h to suppress the Wnt/β-catenin signaling pathway]. The OD values of the TOP flash and the FOP flash were detected by a dual-luciferase reporter assay system (Promega, WI, USA) from cell lysates, and the TOP/FOP ratio reflected the activity of the Wnt/β-catenin signaling pathway.
4
Cyclophosphamide-Induced Chronic Cystitis Model
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Chronic cystitis was induced by systemic intraperitoneal injection of 150 mg/kg of cyclophosphamide (CYP) for 4 consecutive days, based on a previous report [17 (link)]. CYP was dissolved in 0.9% NaCl. Control mice received 0.9% NaCl at the same time. Mice in the model + lenti-NC and model + lenti-Serpina3n groups were intravenously administered with lentiviral particles of lenti-NC and lenti-Serpina3n (5 × 107 pfu per mouse; cat no. MR206624L2V, Origene, Beijing, China) for 3 days prior to cystitis induction, respectively. The model + OE-Serpina3n + XAV-939 group received intraperitoneal injection of XAV-939 (2.5 mg/kg, Selleck Chem, Shanghai, China), an inhibitor of the Wnt/β-catenin signaling pathway, once daily for three times with an interval of 6 h of lentivirus vectors.
5
Wnt Inhibition Enhances Colony Formation
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P2 tdTomato + cells were seeded at a density of 2000 cells per well into 24-well culture plates and left undisturbed for the first 2 days. Then, the growth medium was changed every other day with or without Wnt inhibitor (XAV939) supplementation (1.0 µM, S1180, Selleck, Houston, TX, United States). XAV939 was dissolved in dimethyl sulfoxide (DMSO) according to manufacturer’s recommendations and used in in vitro experiments, and the control group was treated with the same DMSO concentration. After 7 days, the culture plates were stained with a mixture of 0.1% toluidine blue and 2% paraformaldehyde. Colonies with >1 mm diameter were counted as a single colony cluster.
6
Establishing Cancer Cell Lines
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Human lung cancer cell lines (A549, H1299) and a human colorectal cancer cell line (HCT116) were purchased from Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). These cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 10 mg/mL antibiotics (penicillin and streptomycin), and 2 mmol/L L‐glutamine at 37°C under 5% CO2 and saturated moisture. The establishment of cell lines with ING5 stable overexpression or knockdown has been described previously.11 XAV939, ZSTK474, and Niclosamide were purchased from Selleck Chemicals LLC (Houston, TX, USA).
7
Investigating Wnt Signaling in Embryonic Development
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Embryo were obtained by natural mating and cultured in 0.1x MBS to desired stages as described previously30 (link). For in situ hybridization and immunohistochemistry, embryos were fixed at desired stages and processed as described63 . MO 13-3, the antisense MO for ADAM13, was synthesized by Gene Tools, and the sequence was reported previously30 (link). For MO injections, 8-cell stage snai2:eGFP embryos were injected in a single dorsal-animal blastomere with 1.5 ng MO 13-3 using a PLI-100A microinjector (Harvard Apparatus), and Alexa Fluor 555 dextran (Invitrogen) was co-injected as a lineage tracer. For Wnt inhibitor treatment, embryos were cultured in XAV939 or IWR1-endo (both were from Selleckchem) from stage ~28 to ~35. Snai2-eGFP or Wnt reporter transgenic tadpoles were washed three times and subsequently cultured in 0.1x MBS until stage ~44 or ~47 (Fig. 6 ); wild-type tadpoles were immediately fixed and processed for in situ hybridization for snai2 or sox9 (Fig. 7A,B ). For inhibition of Wnt target gene expression, 2-cell stage wild-type embryos were injected in one blastomere with 50 pg mRNA encoding EnR-LefΔN-GR755A, and cultured to stage ~28, when 10 mM DEX (Sigma-Aldrich D4902) or DMSO was added. Embryos were further cultured to stage ~35, fixed, and processed for in situ hybridization for snai2 or sox9.
8
Culturing and Treating GC Cell Lines
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GC cell lines were purchased from the American Type Culture Collection (ATCC) and Korean Cell Line Bank (KCLB). Mycoplasma test was done using MycoAlert™ Mycoplasma Detection Kit (Lonza; LT07-118). Cells were grown in Dulbecco’s modified essential medium or RPMI1640 supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO2. Reagents were sourced commercially as follows: DY131 (#2266; TOCRIS, Bristol, UK), GSK5182 (#AOB1629; Aobious, Gloucester. MA), ICG-001 (#S2662), XAV-939 (#S1180), and Wnt-C59 (#S7037; Selleckchem, Houston, TX), and CHX (#01810; Sigma-Aldrich, St Louis, MO).
9
WNT Signaling Assay in HAP1-7TGP Cells
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To measure WNT reporter activity in HAP1-7TGP cells or derivatives thereof, ~24 hr before treatment cells were seeded in 24-well plates at a density of 8 × 104 per well and grown in 0.5 ml of CGM 2. Cells were treated for 16–24 hr with the indicated concentrations of WNT3A CM, L cell CM, recombinant mouse WNT3A (R and D Systems Cat. # 1324-WN) recombinant human RSPO1 (R and D Systems Cat. # 4645-RS), LiCl, CHIR-99021 (CT99021) (Selleckchem Cat. # S2924) or XAV-939 (Selleckchem Cat. # S1180) diluted in CGM 2. Cells were washed with 0.5 ml PBS, harvested in 150 μl of 0.05% Trypsin-EDTA (0.05%) (Thermo Fisher Scientific Cat. # 25300054), resuspended in 450 μl of CGM 2, and EGFP fluorescence was measured immediately by FACS on a BD LSRFortessa cell analyzer (BD Biosciences) using a 488 laser and 505 LP, 530/30 BP filters, or on a BD Accuri RUO Special Order System (BD Biosciences). Typically, fluorescence data for 5000–20,000 singlet-gated cells was collected and, unless indicated otherwise, the median EGFP fluorescence ± standard error of the median (SEM = 1.253 σ/√n, where σ = standard deviation and n = sample size) was used to represent the data.
10
Cell Line Maintenance and Transfection Protocols
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The human GC cell lines AGS, BGC-823, HGC-27, KATO III, MGC-803, MKN-45, SGC-7901, SNU-1, and NCI-N87 and the human embryonic kidney cell line HEK293T were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were routinely maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37 °C under 5% CO2 in an incubator. XAV-939 (S1180, Selleck, USA), LipofectamineTM 3000 Transfection Reagent (L3000015, Thermo Fisher, USA), riboFECTTM CP Reagent (C10511-1, Ribobio, China), Attractene Transfection Reagent (Cat No. 301005, QIAGEN, Germany), TRIzolTM LS Reagent (Cat No. 10296-028, Thermo Fisher, USA), miRNA mimics (YM00471966-ADA) and inhibitors (YI04100215-DDA, QIAGEN, Germany), the lncRNA FISH probe (lnc1100261, Ribobio, China), a Fluorescent In Situ Hybridization Kit (C10910, Ribobio, China), and h-U6 FISH Probe Mix (lnc110101, Ribobio, China) were used in this study.
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