Wnt3a

Manufactured by Thermo Fisher Scientific

Sourced in United States

Wnt3a is a recombinant protein produced in mammalian cells. It is a member of the Wnt family of signaling proteins and plays a role in various cellular processes, including cell-cell signaling, cell fate determination, and embryonic development.

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59 protocols using wnt3a

Hypoxic Chondrocyte Differentiation of hESCs

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HUES7 hESCs (Howard Hughes Medical Institute/Harvard University, USA) were cultured at 5% O₂ (hypoxia) on Matrigel coated plates as described previously^{40 (link)}. Cells were passaged at ~ 80% confluency using Collagenase IV. hESCs were differentiated into chondrocytes at either 20% O₂, or 5% O₂ in medium (DMEM: F12 containing 1 × non-essential amino acids, 1 × B27 supplement, 90 μM β-mercaptoethanol and 1X ITS supplement: 10 μg/ml insulin, 5.5 μg/ml transferrin and 5 ng/ml selenite premix) supplemented with growth factors over a 14-day period. Medium was replenished daily. Growth factors were added as follows: Day 1, 25 ng/ml WNT3A (R&D) and 50 ng/ml Activin-A (Peprotech); Day 2, 25 ng/ml WNT3A, 25 ng/ml Activin-A and 20 ng/ml FGF2 (Invitrogen); Day 3, 25 ng/ml WNT3A, 10 ng/ml Activin-A, 20 ng/ml FGF2 and 40 ng/ml BMP4 (Peprotech); Days 4–7, 20 ng/ml FGF2, 40 ng/ml BMP4, 100 ng/ml Follistatin (Sigma) and 2 ng/ml NT4 (Peprotech); Day 8, 20 ng/ml FGF2, 40 ng/ml BMP4 and 2 ng/ml NT4; Day 9 and 10, 20 ng/ml FGF2, 20 ng/ml BMP4, 20 ng/ml GDF5 (Peprotech), 2 ng/ml NT4 and 10 ng/ml TGF-β3 (Peprotech); Days 11–14, 20 ng/ml FGF2, 40 ng/ml GDF5, 2 ng/ml NT4 and 10 ng/ml TGF-β3. Cells were dissociated using collagenase IV and passaged at a ratio of 1:2 or 1:3 on days 4 and 9 of the protocol.

Griffith L.A., Arnold K.M., Sengers B.G., Tare R.S, & Houghton F.D. (2021). A scaffold-free approach to cartilage tissue generation using human embryonic stem cells. Scientific Reports, 11, 18921.

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Organoid Infection and Analysis with WNT3A and Nicotinamide

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Freshly split CKIa/p53 DKO and Apc^Min/Min organoids were treated with 80 ng WNT3A (Peprotech) and 10 mM nicotinamide (Sigma-Aldrich) for three days before infection. On the day of infection, organoids were incubated in 200 μl of concentrated virus in infection medium (DMEM/F12 supplemented with WNT3A (80 ng/ml, Peprotech), 10 mM nicotinamide, B27 (1:50, Gibco), mouse Noggin (100 ng/ml, Peprotech), mouse EGF (20 ng/ml, Peprotech), human basic FGF (10 ng/ml, Peprotech) human R-spondin-1 (500 ng/ml, Peprotech), ROCK inhibitor (Sigma-Aldrich) and polybrene (8 μg /ml, Sigma-Aldrich) for 4 h. Infected organoids were collected, Matrigel-embedded and grown in DMEM/F12 medium for five days. Immunofluorescent double staining for p53–GFP and Ki67–GFP was performed and the percentage of organoids with outpocket budding was calculated out of the total GFP-expressing population of organoids. Two independent orga-noid infection cultures were analysed in Fig. 3b. Bright-field and GFP imaging were performed using a fluorescent microscope (Observer Z1, Zeiss).

Kadosh E., Snir-Alkalay I., Venkatachalam A., May S., Lasry A., Elyada E., Zinger A., Shaham M., Vaalani G., Mernberger M., Stiewe T., Pikarsky E., Oren M, & Ben-Neriah Y. (2020). The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic. Nature, 586(7827), 133-138.

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Organoid Infection and Analysis with WNT3A and Nicotinamide

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Adipose-derived Mesenchymal Stem Cell Differentiation

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hAMSCs were isolated from human adipose tissues obtained from donors undergoing liposuction according to our previous studies^{8 (link),30 (link)}. hAMSCs at passage 3 were used in our experiments. All experiments and procedures were approved by the Ethics Committee at the Chinese Academy of Medical Sciences and Peking Union Medical College.
For DE differentiation, as described previously^{8 (link)} and in Supplementary Fig. 1a, hAMSCs at passage 3 were seeded in six-well plates in regular culture medium. The next day, the cells were changed to differentiation basic medium DMEM (Gibco, Grand Island, NY) supplemented with 0.5% FBS (Gibco, Grand Island, NY), 5 ng/ml Activin A (Peprotech, USA), and 50 ng/ml Wnt3a (Peprotech, USA)(AW) or 0.3 µM Chir99021(AC) on the first days, with Wnt3a or Chir99021 being withdrawn on the following 4 days. For the comparison with ESC protocol published by Hannan et al.^{4 (link)} (H), BMP4 (10 ng/ml) and bFGF (10 ng/ml) were added to the differentiation system on the first two days.

Li J., Yang Y., Fan J., Xu H., Fan L., Li H, & Zhao R.C. (2019). Long noncoding RNA ANCR inhibits the differentiation of mesenchymal stem cells toward definitive endoderm by facilitating the association of PTBP1 with ID2. Cell Death & Disease, 10(7), 492.

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Quantifying Wnt-Mediated Transcriptional Activity

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The transcriptional activity of T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) was assayed by firefly luciferase (TOPFlash). Cells were transfected with TOP^Flash reporter or FOP^Flash luciferase reporter and β-gal. β-gal activity was used for normalization. Then, 40 ng/mL Wnt3a (PeproTech, Rocky Hill, NJ, USA) was used for Wnt3a activation. After 72 h, the measurement of luciferase activity was performed, as previously described [14 (link)].

Chen C, & Zhang W. (2019). Itraconazole Alters the Stem Cell Characteristics of A549 and NCI-H460 Human Lung Cancer Cells by Suppressing Wnt Signaling. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 9509-9516.

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Verifying Canonical Wnt Signaling

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In order to verify the activity of canonical Wnt signaling, shCtrl and shIslr C2C12 cells were treated with either Wnt3a (100 ng/ml, Peprotech) or saline for 24 h after 2 d in differentiation medium. The cell samples were then collected for Western blot analysis. In order to activate canonical Wnt signaling in shIslr C2C12 cells, the shIslr C2C12 cells were treated with either 1-azakenpaullone (1-AKP; 3 μM, Sigma) or CHIR-99021 (CHIR; 3 μM, Stemgent) during differentiation. Western blotting and immunofluorescence analysis were performed after 3 or 7 d of differentiation. To verify the activity of canonical Wnt signaling in vivo, the TA muscles of control and Islr cKO mice were injected with Wnt3a (100 ng/ml, Peprotech) or PBS (0.1% BSA) at 1.5 d postinjury. The injured TA muscles were then collected for Western blot analysis at 4 d postinjury. To activate canonical Wnt signaling in vivo, the TA muscles of control and Islr cKO mice were injected with CHIR-99021 (50 ng/ml, Stemgent) or PBS (0.1% BSA) at 2.5 d postinjury. The injured TA muscles were then collected for H&E analysis at 5 d postinjury. To inhibit autophagy, the primary myoblasts of control and Islr cKO mice that we established were treated with bafilomycin A1 (BFA1; 200 nM, Sigma) for 6 h. Then cell samples were collected for Western blot analysis.

Zhang K., Zhang Y., Gu L., Lan M., Liu C., Wang M., Su Y., Ge M., Wang T., Yu Y., Liu C., Li L., Li Q., Zhao Y., Yu Z., Wang F., Li N, & Meng Q. (2018). Islr regulates canonical Wnt signaling-mediated skeletal muscle regeneration by stabilizing Dishevelled-2 and preventing autophagy. Nature Communications, 9, 5129.

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Maintenance and Wnt Activation of Mouse ESCs

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R1 and E14Tg2 mouse ESCs were maintained feeder-free on gelatin- (EmbryoMax 0.1% Gelatin Solution, ES-006-B; Millipore) coated plates in DMEM (41965–039 Gibco), 15% fetal bovine serum (Sigma), 2 mM L-glutamine (25030–024; Gibco), 1X minimal essential medium non-essential amino acids (Gibco), penicillin (100 U/ml) /streptomycin (100U/ml) (15140122; Gibco), 100 μM β-Mercaptoethanol (Gibco) and 1,000 U/ml recombinant mouse leukemia inhibitory factor (ESG1107; ESGRO, Chemicon International). mESCs were treated at indicated concentrations to activate the Wnt pathway: purified Wnt3a (315–20; Peprotech); BIO (361550; Calbiochem); CHIR99021 (361571; Calbiochem). BIO and CHIR99021 were resuspended in DMSO (Sigma) at a stock concentration of 2 mM (BIO) and 6 or 10 mM (CHIR99021), Wnt3a (Peprotech) was resuspended at a stock concentration of 50ng/μL following manufacturer instructions.

De Jaime-Soguero A., Aulicino F., Ertaylan G., Griego A., Cerrato A., Tallam A., del Sol A., Cosma M.P, & Lluis F. (2017). Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus. PLoS Genetics, 13(3), e1006682.

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Reagent Sources for Cell Experiments

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RSL3 was purchased from Fisher Scientific (Cat# 611810). ML210 (Cat#
SML0521), cisplatin (Cat# 1134357), and carboplatin (Cat# C2538) were from
Sigma-Aldrich. WNT3a was from Fisher Scientific (Cat# 5036WN010CF, R&D
Systems), and IWR-1-endo was from Santa Cruz (sc-295215A).

Wang Y., Zhao G., Condello S., Huang H., Cardenas H., Tanner E.J., Wei J., Ji Y., Li J., Tan Y., Davuluri R.V., Peter M.E., Cheng J.X, & Matei D. (2020). Frizzled-7 identifies platinum-tolerant ovarian cancer cells susceptible to ferroptosis. Cancer research, 81(2), 384-399.

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BMP and WNT3A in 3D Cell Culture

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In the assays, either 25–100 ng/ml BMP (R&D SYSTEMS, # 314-BP-050), 50–500 ng/ml WNT3A (Fisher Scientific, # 5036WN010), or the combination of both was added to the culture medium in the Gel-3D culture system and co-culture system for the whole culture time. Dimethylsulfoxide (DMSO; Sigma-Aldrich, # D2650) was added to the control groups.

Esfahani S.N., Zheng Y., Arabpour A., Irizarry A.M., Kobayashi N., Xue X., Shao Y., Zhao C., Agranonik N.L., Sparrow M., Hunt T.J., Faith J., Lara M.J., Wu Q.Y., Silber S., Petropoulos S., Yang R., Chien K.R., Clark A.T, & Fu J. (2024). Derivation of human primordial germ cell-like cells in an embryonic-like culture. Nature Communications, 15, 167.

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Intestinal Crypt Seeding on PLGA Scaffolds

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Caco-2 and HT29-MTX cells were removed from culture flasks with 0.25% (v/v) trypsin, 0.02% EDTA solution in PBS and seeded onto the PLGA scaffolds with a cell concentration of 1 × 10⁶ cells/mL (with a Caco-2/HT29-MTX ratio of 3:1). Media was added to both the basolateral and apical compartments after a 30-min cell attachment period and replaced every 2 days. For studies of basolateral stimulation, media containing epidermal growth factor (EGF; 100 ng/mL, from Invitrogen) was added to the basolateral compartments, with EGF negative media in the apical compartments. Control experiments were performed on cells cultured on standard 0.4 μm pore size 12 mm transwell inserts (Corning Inc., Lowell, MA). Cells were cultured for up to 28 days. PLGA scaffolds were prepared for crypt seeding by coating the scaffold surface with Matrigel^™ (BD Biosciences, San Jose, CA), diluted 1 to 10 in crypt cell media, followed by polymerization at 37°C for 45 min. Intestinal crypt suspensions were then seeded onto the scaffolds for 3 h at 37°C. Unattached crypts were then removed by washing with media, and then samples were incubated with crypt culture media containing added growth factors (500 ng/mL Rspondin, 200 ng/mL WNT3a, 200 ng/mL Noggin, and 50 ng/mL EGF, all from Invitrogen) to enable crypt cell differentiation (Sato et al., 2009 (link)). Crypts were cultured for 7 days on the scaffolds.

Costello C.M., Hongpeng J., Shaffiey S., Yu J., Jain N.K., Hackam D, & March J.C. (2014). Synthetic Small Intestinal Scaffolds for Improved Studies of Intestinal Differentiation. Biotechnology and bioengineering, 111(6), 1222-1232.

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