Recombinant human wnt3a rhwnt3a

Recombinant human Wnt3a (rhWnt3a, 200 ng/mL) was purchased from R&D Systems (Minneapolis, USA). The following antibodies phospho-GSK3β-Ser9 (D85E12), GSK3β (D5C5Z), phospho-Akt-Ser473 (D9E), Akt (C67E7), phospho-β-catenin-Ser552 (D8E11), and β-catenin (D10A8) were all purchased from Cell Signaling Technology (Danvers, USA) and the β-actin antibody from Sigma-Aldrich (Taufkirchen, Germany). The peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Jackson ImmunoResearch (Suffolk, UK).

Human third molars or premolars were obtained from patients (18–25 years old) at The First Affiliated Hospital of Zhengzhou University. All of the donors signed an informed consent form. HDPSCs were isolated and cultivated, as described previously (Gronthos et al., 2000 (link)). Cells from passage 3 and 4 were used for the experiments and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, United States), which was supplemented with 10% fetal bovine serum (FBS; Hyclone, United States), and 100 U/mL penicillin, plus 100 μg/mL streptomycin from (Solarbio, China) at 37°C under 5% CO₂, the medium was changed every 3 days.
Different concentrations, either 0, 50, 100, or 200 ng/mL, of recombinant human Rspo2 (rhRspo2) from PeproTech (United States) were added to the medium for use in subsequent experiments. In addition, 100 ng/mL recombinant human Wnt3a (rhWnt3a) from R&D Systems (United States) or 800 ng/mL recombinant human Dickkopf-related protein 1 (DKK-1; rhDKK-1) from PeproTech (United States) were used in this study.