H3k27me3

Cultured cells in the six-well were extracted on ice using 200 μl of cell extraction buffer (50 mM of Tris-HCl, pH 8.0, 4 M of urea, and 1% Triton X-100) containing protease inhibitor mixture (Roche Diagnostics, 04693132001). Cell lysates were analyzed by Western blotting using EZH2 (CST, 5246, 1:1000), TMEM117 (ProteinTech, 21314-1-AP, 1:500), H3K27me3 (H3K27me3, ab192985, 1:1,000), and GAPDH (ProteinTech, 60004-1-Ig, 1:5,000) antibodies.

The hADSC chondrogenic microspheres and chondrocytes were lysed in radioimmunoprecipitation assay lysis buffer (Cwbio, China). The concentrations of proteins were detected using a bicinchoninic acid kit (Cwbio, China). Protein samples (25 μg) were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, electrophoresis condition: 80 V 25 min, 120 V 60 min; then protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), transfer membrane condition: 250 mA 120 min. The membrane was blocked with 5% skim milk for 60 min. Membranes were incubated with primary antibodies specific for MMP13 (1:1000, Abcam), Col2a1 (1:1000, Abcam), SOX9 (1:1000, Abcam), GPADH (1:000, affinity), KDM6A (1:1000, Proteintech), H3 (1:1000, Proteintech) and H3K27me3 (1:1000, Proteintech). After incubation with a secondary antibody (1:5000, Cell Signaling Technology, Danvers, MA, USA), the signals were detected using a chemiluminescence kit (P90719, Millipore) and chemiluminescence system (Bio‐Rad, Hercules, CA, USA) and analysed using Image Lab Software.