Col10a1

After sequential deparaffinization with xylene and rehydration with graded ethanol, sections were boiled in 10 mM citrate buffer at 95–100°C for 10 min for antigen retrieval, rinsed in 3% H₂O₂ at room temperature for 10 min to inhibit endogenous peroxidase activity, and blocked with 10% goat serum at room temperature for 10 min. Then, the sections were incubated with primary antibodies against collagen 2α1 (COL2A1) (Abcam, 1 : 400) and collagen 10α1 (COL10A1) (Abcam, 1 : 200) at 4°C overnight. After washing, the sections were incubated with secondary antibody at 37°C for 30 min, followed by incubation with streptavidin–HRP conjugate for 20 min at room temperature. Staining without primary antibody was used as a negative control. A microscope (Olympus, Japan) was used for imaging.

After harvesting, total proteins were extracted from the cultures. Protein content was assessed by the Pierce BCA assay kit (Biyuntian, China). Total protein (30 µg) was loaded on to 10% SDS polyacrylamide electrophoresis gels and transferred on to a PVDF membrane. The membrane was immunoblotted overnight at 4°C with antibodies against Col2a1 (1:1000, Abcam, U.S.A.), Sox9 (1:200, Sigma–Aldrich), Runx2 (1:500, Sigma–Aldrich), Mmp13 (1:500, Abcam), Pparγ (1:200, Santa Cruz Biotechnology), Pparγ2 (1:500, Elabscience), Col10a1 (1:200, Abcam), Gapdh (1:2000, Transgen), and β-actin (1:4000, Santa Cruz Biotechnology). The membrane was then incubated with secondary anti-mouse or anti-goat IgG antibodies (1:2000, Transgen) conjugated with peroxidase for 60 min at room temperature. The signal was detected by chemiluminescence using the ECL-Plus Detection System (Transgen). Protein semi-quantitation was based on three independent experiments. The densitometric intensities of protein bands were semi-quantitated using Bandscan 5.0 (Glyko Biomedical, U.S.A.) software and values were normalized to those of β-actin or Gapdh for each sample.

Total proteins were isolated from cells using lysis buffer. The amount of protein was determined using Bradford protein assay according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, USA). Protein samples (20 ug) were separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Bedford, USA). Membranes were incubated with primary antibodies overnight at 4 °C as follows: HDAC1 (1:1000), COL2A1 (1:2000), COL10A1 (1:1000), AGGRECAN (1:1000), COMP (1:1000), MMP-13 (1:1000) (Abcam, Shanghai, China); RUNX2 (1:2000), SOX9 (1:2000), total histone H3 (H3) (1: 5000), acetylated histone H3 (H3_acetyl)(1:2000) (Sigma-Aldrich, Shanghai, China); GAPDH (1:5000). The blots were incubated with secondary antibodies for 1 hour at room temperature. The blots were visualized by SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific™, Waltham, USA) according to the manufacturer's instructions. The intensity of brands was analyzed using Image J [51 ].