Nb100 132

The staining procedure was performed using a Leica Bond III Stainer (Leica,). Slides were subjected exposed to 10 mM sodium citrate buffer, pH 6.0 for 20 min at 37°C. Slides were incubated with the appropriate primary antibody for 15 min, followed by Polymer Refine Detection System (Leica) processing, including hydrogen peroxidase block, secondary antibody polymer, 3,3’ diaminobenzidine and hematoxylin stain. Specimens were then rinsed for 5 min in tap water. Slides were dehydrated in increasing concentrations of ethyl alcohol and xylene prior to permanent coverslipping in xylene-based media. Primary antibodies were as follows: mouse anti-Hif1α (1:400, Novus Biological), mouse anti-HIF2α (Novus Biologicals, NB100-132, rabbit polyclonal, 1:700) mouse anti-H3K9me2 (1:750, Abcam), rabbit anti-H3K27me2 (ab24684, Abcam, 1:500), rabbit anti-5hmC (39769, Active Motif, 1:4000), and mouse anti-SDHB (ab14714, Abcam, 1:1000).

The cell lysates were extracted using RIPA lysis buffer (Beyotime, P0013B) containing 1 mM PMSF (Beyotime, ST505). The samples were heated at 95°C for 5 min in sample buffer containing 2% SDS and 1% mercaptoethanol, separated on 10% SDS‐polyacrylamide gels, and transferred to PVDF membranes by a wet transfer apparatus (Bio‐Rad). The membranes were blotted with 5% BSA for 1 h and then incubated at 4°C. Before incubation with the secondary antibodies, the membranes were washed in TBST solution three times. The following primary antibodies were used: MMP13 (Abcam, ab39012), HIF‐2α (Novus, NB100‐132), GPX4 (Abcam, ab125066), SLC7A11 (Abcam, ab175186), IκBα (Santa Cruz, sc‐1643), NF‐κB p65 (Santa Cruz, sc‐8008) and p‐NF‐κB p65 (Cell Signaling Technology, #3003) and β‐actin (Proteintech, 66009‐1‐Ig). The antibody‐antigen complexes were visualized with Immobilon reagents (Millipore, WBKLS0100).

After fixation, mouse legs were decalcified in 0.5 M EDTA for 2 weeks, embedded in paraffin and sectioned at 4 μm. Slides were stained with safranin O/fast green (Solarbio) using standard protocol. The severity of OA‐like phenotype was analysed using the OARSI scoring system by two blinded observers.
For immunofluorescence analysis, after antigen retrieval and blocking, sections or fixed chondrocytes were incubated at 4°C overnight with primary antibodies against COL2A1 (Abcam, ab34712), MMP13 (Abcam, ab39012), HIF‐2α (Novus, NB100‐132), GPX4 (Abcam, ab125066), NF‐κB p65(sc‐8008) and p‐NF‐κB p65 (Cell Signaling Technology, #3003). The secondary antibodies included donkey anti‐mouse Alexa Fluor 488/555 and donkey anti‐rabbit Alexa Fluor 488/555 (all from Thermo Fisher Scientific). The nucleus was counterstained using 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma, D9542). Images were acquired with a Nicon A1 confocal microscope and processed and analysed with ImageJ software.