Fig 4. UCP2 is Up-Regulated in Sirt1 Knockdown Cells and in Sirt1 KO Mice.

(A) Northern blot for the insulin gene INS-1 in cells with a control vector (pSUPER) or a SiRNA-Sirt1 vector (SiRNA Sirt1). Vertical lines indicate where the respective original blots were spliced together. (B and C) Western blot analyses of targets involved in insulin synthesis and secretion using specific antibodies: cEBP/β, insulin receptor α and β, and kir6.2, one of the K⁺channel receptor subunits. (D) Measurement of ATP/ADP levels in INS-1 control cells (open bars) or Sirt1 knockdown cells (black bars) treated with 16.7 mM glucose (+) or 4 mM glucose (−) (n = 3 experiments done in triplicate, *p < 0.005 in the pSUPER experiment; ANOVA). (E) Western blot analysis for UCP2 in INS-1 control cells (pSUPER) or knockdown cells (SiRNA Sirt1). Vertical lines indicate where the respective original blots were spliced together. (F) Northern blot analysis for UCP2 in INS-1 control cells (pSUPER) or knockdown cells (SiRNA Sirt1). Vertical lines indicate where the respective original blots were spliced together. (G) NADH levels in INS-1 cells after glucose addition as determined by autofluorescence [46] and expressed as arbitrary units. Cells stably transfected with control or Sirt1 SiRNA vectors were used (n = 2, *p < 0.05 compared with no glucose). (H) UCP2 protein levels in isolated pancreatic islets of two wild-type or two Sirt1 KO mice. Tubulin or actin was used as loading control in all Western and Northern blots.