Foxa1

Cells were harvested with 1× cell lysis buffer (Promega). A total of 60 μg of total proteins were separated on 10% polyacrylamide gel and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 1% bovine serum albumin (BSA) in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated with antibodies against FOXA1, BCL-2, CyclinD1 (Proteintech) or GAPDH (#2118, Cell Signaling Technology) overnight at 4°C. After extensive washing with TBST buffer, the blots were then incubated with goat anti-rabbit, horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescence reagents ECL (Millipore, MA, USA) and the intensity of the bands was analyzed using Image J software (National Institute of Health, USA).

HBMSCs and mBMSCs were provided by Cyagen Biosciences (HUXMA-01001, MUBMX-01001, Guangzhou, China), which can differentiate into osteoblasts, chondrocytes, and adipocytes under specific inductive conditions. Adherent hBMSCs were incubated in culture flasks in a special complete growth medium (HUXMA-90011, Cyagen Biosciences, Inc., Guangzhou, China) in a cell incubator at 37 °C with 5% CO₂ and were passaged at nearly 80–90% confluence. Cells from passages two to six were used in subsequent experiments.
Specific antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and FOXA1 were purchased from PROTEINTECH (Chicago, USA). Runt-related transcription factor 2 (RUNX2), extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2) were obtained from Cell Signalling Technology (Danvers, MA, USA). Specific antibodies against collagen type I alpha 1 (COL1A1) and Osterix (SP7) were purchased from Abcam (Cambridge, UK) and PROTEINTECH (Chicago, USA). A phospho-p44/42 MAPK (P-ERK1/2) inhibitor, PD98059, was purchased from MedChemExpress (New Jersey, USA).