Col2a

The de-paraffinized knee joint sections from DMM and sham mice were incubated overnight at 4° C with primary antibodies against BMP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Runx2, Col2a, MMP13, Adamts5 (Abcam, Cambridge, UK), as well as p16 and p21 (Proteintech, Rosemont, IL, USA). The slides were then incubated with the secondary antibody (Jackson ImmunoResearch Laboratories, Baltimore Pike, USA, 1:200)) in blocking solution for 1 h at room temperature, and developed with 3,3′-diamino-benzidine (DAB, ZSGB-Bio, Beijing, China).

CEP tissue protein and endplate chondrocytes protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to apolyvinylidene fluoride (PVDF) membrane. The samples were sealed with 5% skim milk, and the PVDF membrane was placed in the following primary antibodies: YAP1(1:2000; Cell Signaling Technology), Sox9(1:1000; Affinity), COL-2A(1:1000; Abcam), p53(1:1000; Abclonal), p21(1:1000; Affinity), connective tissue growth factor (CTGF, 1:1000; Affinity), and β-actin (1:1000; Zs-BIO). The PVDF membrane was then placed into the secondary antibody solution and incubated for 1 h. The protein was imaged through a gel imaging system in the presence of an ultra-sensitive luminescent chromogenic solution. Finally, the intensity of the bands was quantified using image grayscale analysis.