Figure 3

cAMP Promotes Sirtuin to Prevent Oxidative Damage and Cell Senescence.

(A) Cell cycle detection by PI staining. (B) Fluo-3 staining for C2C12 cells. (C) Western blot results for the CaMKKII, SIRT1 and SIRT3 protein levels with or without the CaMKKII specific inhibitor STO609. cAMP, db-cAMP and Rolipram were also added (100 μM). (D) Western blot results for the AMPK, SIRT1 and SIRT3 protein levels with or without the AMPK-specific inhibitor Compound C. cAMP, db-cAMP and Rolipram were also added (100 μM). (E) Western blot results for the PKAc, SIRT1 and SIRT3 protein levels with or without the PKA-specific inhibitor H89. cAMP, db-cAMP and Rolipram were also added (100 μM). All of the gels (C–E) were run under the same experimental conditions. (F) Fold change of cAMP levels in cells treated with cAMP, db-cAMP (10, 100, 1000 μM) and Rolipram (100 μM). (G) Differentiation of C2C12 cells with or without db-cAMP or Rolipram treatment. Blue points indicate the nucleus of cells stained with Hoechst 33342. (H) SA-β-gal staining for C2C12 cells with or without H₂O₂ treatment. db-cAMP and Rolipram added at different concentrations (10, 100, 1000 μM). All the statistical analyses were performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated control group cells.