Figure 2. SIRT6 activity is altered by age and oxidative stress.

(A) Unstimulated confluent primary human chondrocytes isolated from young (42.14±2.8 years) and old (70.85±5.6 years) cartilage were lysed and histones were extracted prior to immunoblotting for the acetylated form of H3K9 (H3K9ac) as an inverse marker of basal SIRT6 activity. Representative immunoblots from young and old chondrocytes are shown. (B) Densitometric analysis showing basal H3K9ac protein levels in young and old chondrocytes (n=7). H3K9ac was normalized to total histone 3 as a loading control. Unpaired t-test was used to test for statistical differences between age groups. (C) Recombinant human SIRT6 was treated in the presence or absence of 50 μM H₂O₂ or the SIRT6 inhibitor, EX527 (100 μM), for 15 mins and a deacetylation-based assay was used to measure recombinant SIRT6 activity. Data are expressed as percentage SIRT6 activity compared to control (recombinant SIRT6 alone) at 15 mins (n=3 independent experiments). One-way ANOVA was used to test for differences compared to control. (D) Chondrocytes were treated with varying concentrations of exogenous H₂O₂ (0-50 μM) for 30 mins and biotinylated IAM (BIAM) was used to affinity tag proteins containing a variety of thiol oxidative modifications (R-SOH, R-SS-R, R-SSG, R-SNO) followed by pull-down with streptavidin beads and immunoblotting for SIRT6. (E) Chondrocytes were treated with FN-f (1μM), menadione (25 μM) or DMNQ (25 μM) for 30 mins, and DCP-Bio1 was used to specifically affinity tag proteins containing R-SOH (cysteine sulfenic acid intermediate) followed by pull-down with streptavidin beads and immunoblotting for SIRT6 and Prx2. Data shown are representative blots from n=3 independent experiments. AhpC was used as a loading control in BIAM and DCP-Bio1 experiments. (Mena.; menadione). (F) Human chondrocytes were transduced with an empty vector control (ad-Null) or an adenoviral vector encoding SIRT6 (ad-SIRT6) for 48 hours. A representative immunoblot showing typical levels of SIRT6 overexpression from total cell lysates is shown along with quantification from n=27 donors. (G) Chondrocytes were treated with 25 μM menadione for 0-30 mins to induce oxidative stress. Histones were extracted and immunoblotting for H3K9ac was used to assess SIRT6 activity levels. Representative immunoblots are shown. (H) Densitometric analysis from n=3 independent samples. H3K9ac protein levels were normalized to total histone 3 as a loading control. Asterisks represent significant differences compared to controls (**, p<0.01). Two-way ANOVA was used to test for differences between highlighted groups.