Col2a1

The drug-treated chondrocytes were lysed using a mixture of radio immunoprecipitation assay (RIPA) lysis buffer and protease inhibitor cocktail. The proteins with equal amounts (30 μg) were separated on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% bovine serum albumin for 1 h, the membranes were incubated with the primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with the secondary antibodies for 1 h at ambient temperature. Lastly, the membranes were visualized using the BIO-RAD ChemiDoc XRS+system. The primary antibodies consisted of COL2A1 (Bioss, 1 : 1000), ACAN (Bioss, 1 : 1000), FoxO1 (CST, 1 : 1000), FoxO3a (CST, 1 : 1000), p-FoxO3a (CST, 1 : 1000), FoxO4 (CST, 1 : 1000), PI3K (CST, 1 : 1000), p-PI3K (CST, 1 : 1000), AKT (CST, 1 : 1000), p-AKT (CST, 1 : 1000), Bax (CST, 1 : 1000), Bcl-2 (CST, 1 : 1000), Caspase 3 (CST, 1 : 1000), SOD2 (ABclonal, 1 : 1000), CAT (ABclonal, 1 : 1000), GAPDH (CST, 1 : 1000), and β-actin (CST, 1 : 1000).

The paraffin-embedded tissue sections were deparaffinized and rehydrated following standard procedures. Sections were incubated with 3% H₂O₂ to block endogenous peroxidase activity and antigen retrieval was performed in citrated buffer at 110 ℃, for 5 min in a pressure cooker. After the citrated buffer reached room temperature, the sections were removed and incubated overnight with the primary antibodies COL2A1 (1:200, bioss, bs-10589R) and SOX9 (1:1000, Abcam, Cat# ab185966) at 4 ℃, followed by incubation with an HRP conjugated secondary antibody (Beyotime Institute of Biotechnology, Inc., Nantong, China) for 2 h at room temperature. Peroxidase binding for both COL2A1 and SOX9 was visualized using diaminobenzidine. Then, the nuclei were counterstained with hematoxylin, while the slides were dehydrated, mounted, and analyzed with a light microscope. For the quantitative analysis, all positively stained cells, including those in the femoral condyle and tibial plateau area, on the articular surface per specimen were counted, and the percentage of positive cells was calculated using Image-Pro Plus 6.0.

Cells were washed with phosphate-buffered saline (PBS) and harvested in radioimmunoprecipitation assay buffer (Beyotime, Haimen, China) supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of protein were loaded on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was probed with specific antibodies overnight at 4 °C and was then incubated with secondary antibodies at room temperature for 1 h. Each protein band was visualized with an ECL reagent (Millipore). Primary antibodies specific for the following proteins were used: Bax, Bcl-2, cleaved Caspase 9, cleaved PARP1, and Survivin (all from Proteintech Group, Wuhan, China), cleaved Caspase 3 (Affinity, Cincinnati, OH, USA), ANKH (ABclonal, Wuhan, China), AQP1, β-actin, MMP13, COL2A1, and ADAMTS-5 (all from Bioss, Beijing, China).