Wnt7b

For WNT ligands treatment during chondrogenesis, pellets were treated with either 10 ng ml⁻¹ TGF-β3 (control group) or a combination of 10 ng ml⁻¹ TGF-β3 and 100 ng ml⁻¹ individual WNT ligand (WNT2B, WNT3A, WNT4, WNT5B, or WNT7B, all from R&D system) in chondrogenic medium from d0 to d42 as appropriate. For WNT ligands treatment during the Cp stage, cells were supplemented with either 20 ng ml⁻¹ BMP4 (R&D Systems, #314-BP-010) alone (control group), a combination of 20 ng ml⁻¹ BMP4 and 1 μM C59, or a combination of 20 ng ml⁻¹ BMP4 and 100 ng ml⁻¹ WNT3A (R&D Systems, #5036-WN-010) in mesodermal differentiation medium from d7 to d12.

Cells were lysed using cold RIPA lysis buffer (Beyotime), and lysates were centrifuged at 10000 × g for 30 min at 4℃, retaining the supernatants. The total protein concentration was quantified using a BCA Protein Assay Kit (Beyotime, Institute of Biotechnology). Total proteins were loaded on an 8% SDS‐gel, resolved using SDS‐PAGE, and then transferred to a PVDF membrane. Membranes were blocked in 5% non‐fat dried milk in TBST for 2 h and then incubated with the specific primary antibodies overnight, using GAPDH as the loading control. Membranes were subsequently incubated with horseradish peroxidase‐coupled secondary antibody for 2 h. The gray value of bands was normalized to the respective GAPDH bands. The primary antibodies used were as follows: Fzd7 (1:1000; Abcam, cat. no. ab64636), Wnt7b (1:1000; R&D Systems, cat. no. AF3460), ABCG2 (1:1000; Cell Signaling Technology, Inc.; cat. no. 42078), β‐catenin (1:1000; Cell Signaling Technology, Inc.; cat. no. 9562), active β‐catenin (1:1000; Cell Signaling Technology, Inc.; cat. no. 8814), and GAPDH (1:10,000; ProteinTech Group, Inc.; cat. no. HRP‐60004).