FIGURE 2.

Transient changes in SirT1 levels or activity alters cartilage gene expression. OA human chondrocytes (P0) were transiently transfected with a SirT1 expression plasmid. Extracts generated at 3 days post-transfection were used in immunoblot analysis (A) or SirT activity assays (B). C, RNA isolated from the transfected cells in A was used in RT-PCR assays with the indicated human primers. The graphs show relative induction of gene expression (compared with control). D, P0 and P1 OA chondrocytes were treated with resveratrol (1 μm) for 3 days at which time RNA was isolated and used in RT-PCR reactions with the indicated human primers. E, OA chondrocytes (P0) were transiently transfected with a SirT1 siRNA, and extracts were isolated and used in immunoblots for SirT1 and GAPDH (upper panel) or for assessment of SirT activity (lower panel). CTL, control. F, RNA was isolated from the cells expressing the SirT1siRNA in E which was used in RT-PCR reactions with the indicated human primers. G, OA chondrocytes were treated with NAM (10 mm) for 3 days at which time total RNA was isolated and used RT-PCR reactions with the indicated human primers. The graphs in F and G show relative repression of gene expression. The error bars in the graphs indicate the S.D., and the statistical significance is indicated by an asterisk (^*) (LSD, p < 0.05). The displayed immunoblots are representative of four experiments. RT-PCR was performed in triplicate for two experimental repetitions (n = 6).