Western blotting was performed as previously described (25 (link)). Cell lysis was performed in a buffer containing 0.25 M Tris-HCl, 20% glycerol, 4% sodium dodecyl sulfate (SDS) and 10% mercaptoethanol (pH 6.8) supplemented with protease and phosphatase inhibitors. The protein contents were measured using the Micro BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of total protein (10 µg) were separated on 10-12% SDS-polyacrylamide gels and electro-transferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h, the membranes were incubated with primary antibodies (1:3,000; collagen-II, cat. no. ab34712, Abcam; aggrecan, cat. no. ab194594, Abcam; MMP-2, cat. no. ab97779, Abcam; GAPDH, cat. no. ab9485, Abcam) in TBST containing 5% non-fat milk overnight at 4°C. Secondary horseradish peroxidase‑conjugated antibodies (1:6,000; Goat Anti-Rabbit IgG H&L, cat. no. ab6721; Goat Anti-Mouse IgG H&L, cat. no. ab205719, Abcam) were added at room temperature for 1 h, and immunoblots were developed using an enhanced chemiluminescence system (Cytiva). ImageJ 1.47 (National Institutes of Health) was used for densitometry analysis.