Figure 3. Identification of Sirt3-regulated acetylation sites in human cells.

(A) Generation of Sirt3 knockdown and overexpression U2OS cells. U2OS cells were either transfected with a human Sirt3 encoding cDNA or with a Sirt3-specific shRNA. To induce expression of Sirt3-shRNA, cells were treated with doxycycline for the indicated time periods. Expression of Sirt3 was analyzed by immunostaining using anti-Sirt3 antibody. (B) Distribution of identified acetylation sites between mitochondrial or non-mitochondrial cellular compartments. (C) Increased acetylation of mitochondrial acetylation in Sirt3 knockdown cells. Logarithmized SILAC ratios of acetylated peptides from mitochondrial and non-mitochondrial proteins were plotted. These data shows the upwardly shifted distribution of SILAC ratios of mitochondrial acetylation sites in Sirt3 knockdown cells. (D) Gene Ontology enrichment analysis of Sirt3-regulated proteins. Proteins with increased acetylation in Sirt3 knockdown cells showed significant enrichment of mitochondrial associated GO terms.