Osterix

Paraffin-embedded sections were mounted on slides, dewaxed, and dehydrated. After washing, slides were incubated with primary antibodies against platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31, Affinity, Changzhou, Jiangsu, China), runt-related transcription factor (RUNX2, Affinity, Changzhou, Jiangsu, China), osterix (Affinity, Changzhou, Jiangsu, China), and type I collagen (Affinity, Changzhou, Jiangsu, China) overnight at 4°C, respectively. After washing with PBS, slides were incubated with a horseradish peroxidase-conjugated secondary antibody (Thermo Fisher, Cambridge, MA, USA) for 60 min at 37°C. Then these slides were stained with DAB (Solarbio, Beijing, China) and counterstained with hematoxylin (Solarbio, Beijing, China).

The total protein in the cells was extracted using RIPA lysis buffer, and the concentration of the protein sample was determined using a BCA kit (Beyotime Biotechnology). Each sample underwent a 10‐min denaturing process at 100°C. Samples were separated using a 10% SDS polyacrylamide gel (Beyotime Biotechnology) and transferred to PVDF membranes (Millipore). The membrane was first blocked with 5% nonfat milk TBST buffer for 2 h, and primary antibodies were applied and incubated overnight at 4°C, followed by the application of secondary antibodies for 1 h. Blots were visualized using BeyoECL Moon (Beyotime Biotechnology). The antibodies used in this study included Fas (1:2000; R&D System), FasL (1:1000; Bioss), COL‐I (1:1000; Immunoway), integrin β1 (1:1000; Immunoway), Fibronectin (1:1000; Immunoway), RUNX2 (1:1000; Immunoway), Osterix (1:1000; Affinity), ALP (1:2000; Santa Cruz Biotechnology), LC3B (1:1000; Abcam), Beclin (1:2000; Santa Cruz Biotechnology), HRP‐conjugated secondary antibodies against rabbit and mouse (1:10000; Biosharp), and HRP‐labelled donkey anti‐goat IgG (1:1000; Beyotime).