Trf 78

To detect DNA damage and telomere dysfunction, the γH2AX foci and TIFs were analyzed 48 h after lipofection. In brief, human chondrocytes were seeded onto coverslips and cultured for 24 h at 37 °C in an incubator under 20% O2/5% CO₂. After chondrocytes had been cultured for 24 h, pcDNA3.1-Sirt6, siRNA Sirt6, or corresponding controls were transfected by lipofection. Forty-eight hours after lipofection, chondrocytes were fixed for 10 min with 4% paraformaldehyde in PBS, followed by permeabilization with 0.25% Triton X-100 in PBS for 10 min. The cells were subsequently washed with PBS, blocked for 1 h with 1% BSA (Sigma-Aldrich) in PBS containing 0.1% Tween-20, and then incubated with an anti- TRF-1 mouse monoclonal antibody (TRF-78, ab10579, Abcam, 1:1000 dilution). γH2AX was detected by incubation with a rabbit polyclonal antibody (Phospho-Histone H2A.X Ser139, Cell Signaling, 1:100 dilution, #9718).