For the luciferase assay, cells were seeded on a 24-well culture plate and grown overnight. The Topflash reporter plasmids were co-transfected with the Renilla construct (0.01 µg) and AKAP2 expression vector with Lipofectamine 2000 to assess the transcriptional activity of β-catenin/TCFcomplex. After incubation for 48 h, cells were treated with Wnt3a protein (10 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA) for 8 h. Then, the transfected cells were washed twice with phosphate-buffered saline (PBS) and total proteins were lysed in passive lysis buffer (100 µl/well) provided by the Dual-Luciferase Reporter Assay system kit (Promega Corporation, Madison, WI, USA). Firefly and Renilla luciferase activities were measured from the lysate using a Centro LB 960 Microplate Luminometer (Berthold). Firefly luciferase values were normalized to Renilla luciferase values to control for transfection efficiency.
Wnt3a protein
Manufactured by R&D Systems
Sourced in United States
Wnt3a protein is a signaling protein that plays a key role in the Wnt signaling pathway. The Wnt3a protein is involved in various cellular processes, including cell fate determination, cell proliferation, and cell migration. It is an important regulator of embryonic development and tissue homeostasis.
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Lab products found in correlation
Wnt5a protein, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Centro lb 960 microplate luminometer, by Berthold Technologies (1 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Helistat collagen sponge, by Integra LifeSciences (1 mentions)
Sfrp1, by R&D Systems (1 mentions)
Stemi 2000, by Zeiss (1 mentions)
Mccoy 5a media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Stempro accutase cell dissociation reagent, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
7 protocols using wnt3a protein
1
Wnt-Dependent Transcriptional Assay
2
Wnt Signaling Pathway Modulation
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cWnt signaling activation and inhibition animal surgical procedures were conducted as previously described13 (link). A skin incision was performed above the PF or SAG suture of anesthetized pN4 CD-1, Axin2LacZ/+, or ActinCreERT2:Rainbow mouse under the aid of a dissection stereo microscope Stemi 2000 (Zeiss, Oberkochen Germany). A 1.5 mm diameter Helistat collagen sponge (Integra LifeSciences, Plainsboro, NJ, USA) was either soaked in 150 ng of Wnt3a protein (R&D Systems, Minneapolis, MN, USA) and placed on the PF suture, or with a combination of 2 μg of Dkk-1 and 2 μg sFrp-1 (R&D Systems, Minneapolis, MN, USA) and placed on the SAG suture. PBS soaked collagen sponges were used as controls in both sutures. The incision was closed using a single 8-0 nylon suture and the animals were allowed to recover. This procedure was repeated every other day till pN8 or pN17 specifically, the incision was re-opened, the collagen sponge re-soaked with the corresponding factors. On day pN8 or pN17 animals were sacrificed and processed for histological or FACS analysis. For information regarding reagents used refer to Supplementary Table 4 .
3
Wnt Activation in Colorectal and Kidney Cell Lines
4
Isolation and Expansion of Skeletal Stem Cells
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Skeletal stem/progenitor cells were isolated from entire cranial and tibias from pN3-5 wild type (C57BL/6) or EGFP+/− mice by flow cytometry. Skeletal stem/progenitor cells were pelleted at 1350 rpm at 4 °C for 20 min, resuspended in growth media (as described above) supplemented with 50 ng/ml Wnt3a protein (R&D Systems, Minneapolis, MN, USA) and seeded at density of 3 × 105 cells/well into a 96-well plate. Cells were incubated under standard conditions (37 °C, 5% CO2) overnight (8–12 h). The following morning, cells were lifted using StemPro Accutase Cell Dissociation Reagent (Gibco-Life Technologies, Grand Island, NY, USA), pelleted at 1350 rpm for 20 min at 4 °C. Pelleted cells were then resuspended in 2 μl of recombinant Wnt3a protein at concentration of 100 ng/μl in PBS for transplantation. For information regarding reagents used refer to Supplementary Tables 3 and 4 .
5
Wnt5a and Wnt3a Effects on Cell Cycle
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JB6 cells were cultured in DMEM (Hyclone, USA) containing 10% FBS (Gibco, USA). For cell cycle analysis, JB6 cells were plated onto 6-well plate at a confluence of 70% to 80%. After 6 hours, Wnt5a protein (4 μg, R&D, USA), Wnt3a protein (1 μg, R&D, USA) or control dissolvent was added to the culture medium. Cells were harvested after 24 hours, and cell cycles were analyzed under a fluorescence-activated cell sorter according to standard protocols.
6
MTT Assay for Wnt Protein Effects
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JB6 cells were plated onto 96-well plates. Six hours later, Wnt5a protein (0.2 μg, R&D, USA), Wnt3a protein (0.1 μg, R&D, USA) or control dissolvent were added to the culture medium. Twenty-four hours later, 20 μl of 5 mg/ml MTT was added to each well. Four hours later, the culture medium was aspirated out, 150 μl DMSO (Invitrogen, USA) was added, and the plates were shaken for 10 minutes. The absorbance value was determined by an enzyme-linked immunoassay at 490 nm. Significant differences were evaluated using a t-test, and p< 0.05 was considered statistically significant.
7
Murine and Human Osteosarcoma Cell Culture
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Murine osteosarcoma cell line LM8 (Riken Cell Bank, Tokyo, Japan, RCB Cat# RCB1450, RRID: CVCL_6669) was cultured in α-MEM containing 10% fetal bovine serum (FBS). Human osteosarcoma cell line 143B (Riken Cell Bank, RCB Cat# RCB0701, RRID: CVCL_9W36) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% FBS. All cell cultures were maintained in 5% CO2 at 37 °C. Recombinant mouse and human SOST/Sclerostin protein (Cat# 1589-ST and 1406-ST) and Wnt3a protein (Cat# 1324-WN and 5036-WN) were purchased from R&D Systems Co. Ltd. (Minneapolis, MN, USA).
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