Research Paper Volume 10, Issue 5 pp 1053—1072

Deacetylation of metabolic enzymes by Sirt2 modulates pyruvate homeostasis to extend insect lifespan

Figure 5. Sirt2 interacts with and stabilizes PEPCK levels. (A) Sirt2 directly binds to PEPCK. HzAm1 cells were co-transfected with GFP-Sirt2 and GFP-PEPCK-V5 plasmids for 48 h, and the cell extracts were immunoprecipitated (IP) with anti-V5 or anti-Sirt2 antibody, followed by immunoblotting (IB) with anti-Sirt2 or anti-V5 antibody, respectively. WCE: whole cell extracts. (B) Sirt2 transfection increases endogenous PEPCK protein levels in vitro. (a) Dose-dependent response to Sirt2 overexpression. HzAm1 cells were transfected with GFP-Sirt2 or GFP-V5 plasmid for 48 h. (b) Time-dependent response to Sirt2 transfection. HzAm1 cells were transfected with 1.5 μg of recombinant Sirt2 plasmid. (C) Sirt2 knockdown decreases endogenous PEPCK protein levels in vitro. (a) Dose-dependent response to Sirt2 RNAi. HzAm cells were transfected with Sirt2 or GFP dsRNA for 48 h. (b) Time-dependent response to RNAi. HzAm1 cells were transfected with 4 μg Sirt2 dsRNA. (D) Effects of Sirt2 inhibitor AGK2 on PEPCK protein levels. (a) Dose-dependent response to AGK2 treatment. HzAm1 cells were cultured with AGK2 for 48 h. (b) Time-dependent response to AGK2 treatment. HzAm cells were cultured with 10 μM AGK2. Proteins were extracted from the cells for IB with anti-PEPCK antibody. Protein bands were quantified using ImageJ software and normalized to the levels of H. armigera actin (5 μg). Each point represents the means±S.D. of three independent replicates. *, p<0.05; **, p<0.01 (determined by independent t-test).