Western blot analysis

Chondrocytes were washed three times with ice-cold PBS and total protein was extracted using 0.2 ml RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Chondrocytes were incubated in lysis buffer for 30 min on ice followed by centrifugation at 18,894 × g for 20 min at 4°C. Total protein was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) and 25 µg protein was separated via SDS-PAGE on a 6 or 10% gel. The separated proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA) and blocked for 1 h at 25°C with Tris-buffered saline containing 0.1% Tween™ 20 and 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) to prevent non-specific binding. The membranes were incubated with mouse primary antibodies against NF-κB1 (1:5,000; cat. no. MA5-15128), MMP-13 (1:5,000; cat. no. MA5-14247), caspase-3 (1:5,000; cat. no. MA1-91637), IL-6 (1:5,000; cat. no. M621B all Invitrogen; Thermo Fisher Scientific, Inc.) or β-actin (1:10,000; cat. no. 3700S; Cell Signaling Technology, Inc., Danvers, MA, US) for 12 h at 4°C. Following primary incubation, membranes were incubated with anti-mouse horseradish peroxidase (HRP)-labeled secondary antibodies (1:12,000; cat. no. 7076S; Cell Signaling Technology, Inc.) at room temperature for 1 h. Protein bands were visualized using RapidStep™ ECL detection reagent (EMD Millipore) and Syngene GeneGenius Gel Light Imaging system (Syngene, Frederick, MD, USA), according to the manufacturer's protocol. Each experiment was performed in triplicate.