Figure 6.

Sirt1 Inhibits iNOS Expression in Inflammatory Cytokine-Induced MSCs through Deacetylating p65

(A) MSCs were pretreated with or without NF-κB inhibitor SN50 (18 μM) for 2 h, and then MSCs were stimulated with TNF-α and IFN-γ (IT, 10 ng/mL, each) for 24 h. MSCs treated without SN50 and IT were used as the control. After 24 h, cells were subjected to western blot for analyzing iNOS protein level. GAPDH was used as the internal control. (B) Under the same treatment conditions as in (A), the nitrite level in cell culture supernatant was measured using Griess reagent.^. **p < 0.01 versus IT + MSC; ***p < 0.001 versus control MSC. (C) AdEGFP-MSCs or AdSirt1-MSCs were treated respectively with IT (10 ng/mL, each) for 30 min, and MSCs treated with IT (10 ng/mL, each) for 30 min were used as the control. The cells were then subjected to western blot for analyzing protein levels of acetyl-(Lys310)-p65, p65, and Sirt1. GAPDH was used as the internal control. (D) MSCs, AdEGFP-MSCs, or AdSirt1-MSCs were treated respectively with IT (10 ng/mL, each); after 30 min, proteins were extracted for immunoprecipitation with anti-p65, and the immunoprecipitate was subjected to immunoblotting (IB) with antibodies against acetyl-NF-κB p65 (Lys310) antibody.