Recombinant mouse wnt 3a protein

To further verify whether SR regulates osteoblasts via the WNT/β-catenin signal pathway, the WNT signaling was activated or inhibited. For activation, 5 ng/ml recombinant mouse Wnt-3a protein (R&D systems) was added to the above-described culture system of MC3T3-E1, and the luciferase activity of cells was measured after 48 h of co-treatment with SR. For inhibition, 5 μM ICG-001 (MedChemExpress), which specifically binds to cyclic AMP response element-binding protein, was used to antagonize WNT/β-catenin/TCF transcription [31 (link)]. The inhibitory treatment and cell luciferase activity assay were identical to the activation treatment by Wnt-3a.

The next day, cells were transferred from PMEA chamber slide to Matrigel-coated culture plates. Cells in the chamber slide were washed twice with PBS, and then overlaid with Gibco TrypLE^TM Express (Thermo Fisher Scientific), and incubated at 37 °C for 10 min. Cells were collected in standard medium supplemented with 10% FBS, and then centrifuged at 300× g for 5 min. After discarding the supernatant, cells were recovered with modified ES medium [48 (link)] and seeded on Matrigel (Corning, NY, USA)-coated 24- or 96-well plates. Fresh ES medium was added every 2 or 3 days.
The modified ES medium contained Advanced DMEM/F12 (Thermo Fisher Scientific), Penicillin-Streptomycin Solution Hybri-Max^TM (100 ng/mL; Sigma Aldrich, MO, USA), gentamycin sulfate solution (25 mg/mL, Wako Pure Chemical, Osaka, Japan), HEPES buffer solution (10 mM; Thermo Fisher Scientific), B-27 supplement (Thermo Fisher Scientific), bovine serum albumin (1 mg/mL; Sigma Aldrich), Glutamax (Thermo Fisher Scientific), recombinant murine Noggin (50 ng/mL; Pepro Tech, Cranbury, NJ, USA), recombinant human R-Spondin1 (500 ng/mL; R&D Systems, Minneapolis, MN, USA), recombinant murine EGF (50 ng/mL; Pepro Tech), recombinant mouse HGF protein (50 ng/mL; R&D Systems), recombinant mouse Wnt-3a protein (100 ng/mL; R&D Systems), Culture Sure Y-27632 (10 μM; Wako), and hydrocortisone (0.5 μM).