8 ohdg

High‐glucose (25 mM) Dulbecco's modified Eagle's (DMEM‐H), foetal bovine serum (FBS), antibiotics (penicillin and streptomycin), and Lipofectamine2000 were obtained from Thermo Fisher Scientific. Methyl viologen dichloride hydrate (paraquat, PQ), dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA), DAPI, acridine orange (AO), dansylcadaverine (MDC), and NAO dye were purchased from Sigma (MO, USA). Rapamycin (RAPA) and 3‐methyladenine (3‐MA) were purchased from Selleckchem. ST2825 was purchased from MedChemExpress. All molecular biological reagents were obtained from New England Biolabs. Primary antibodies against mTOR, phosphor(P)‐mTOR, P‐p53, Drp1, Tom20, cleaved‐caspase 3, and Bcl‐2 were purchased from Cell Signalling Technology. Anti‐Ki67, phosphorylated histone H2A.X (P‐γH2A.X), CD3, and CD68, and Alexa Fluor® 488‐conjugated or Alexa Fluor® 594‐conjugated second antibody were purchased from Abcam. Anti‐LC3B, SQSTM1/P62, and MyD88 were purchased from Sigma. Anti‐Beclin 1, CDK2, GPX1, 8‐OHdG, OPA1, and Fis1 were purchased from Bioss (Woburn, MA, USA). Anti‐SOD1, p53, PARP1, NDUFB8, and UQCRFS1 were purchased from Proteintech. Secondary antibodies horseradish peroxidase‐conjugated goat anti‐rabbit or mouse IgG was purchased from ZSGB‐Bio.

Immunostaining for COL2 (Abcam), MMP13 (Abcam), 8-hydroxy-2'-deoxyguanosine (8-OHdG, Bioss), type 1 collagen/COL1 (Abcam), NOX4 (Abcam) (Rabbit, 1:100 for each antibody), AGG (Bioss) (Rabbit, 1:50) and F4/80 (Abcam) (Rat, 1:200) was performed on decalcified paraffin-embedded sections of the operated knees. Heat-induced antigen retrieval involved incubation with citrate buffer (pH 6.0) at 80°C for 20 min. Sections were then blocked with peroxidase blocking solution (Dako) for 5 min. Non-specific binding sites were blocked with 5% normal goat serum in 0.3% Triton X100 (Sigma-Aldrich) for 60 min. Primary antibodies were incubated at 4°C overnight. Rabbit (Dako) or rat HRP-conjugated secondary antibodies were incubated for 30 min at room temperature and revealed with the DAB Substrate Kit (Abcam).
To investigate the role of NOX4 and the effect of inflammation on cartilage, untreated and IL-1β-treated (72 hours) cartilage explants of WT and NOX4^-/- mice were fixed with paraformaldehyde, embedded in OCT, and stained with safranin-O or analysed by immunohistochemistry. Cryosectioned slides were subject to immunostaining with the antibodies against COL2, COL1, 8-OHdG, NOX4, AGG and MMP13 (1:200 for each antibody) using the protocol described above. Quantification was performed by manually counting the number of positive staining cells using the FIJI software.