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. 2022 May 19;53:102334. doi: 10.1016/j.redox.2022.102334

Fig. 1.

Fig. 1

SIRT1 inhibition promotes mitochondrial acetylation. (A) Western blot analysis of lysine-acetylation (K–Ac) expressions in whole-cell lysates of HCT116 and DLD1 cells treated with increasing concentrations of INZ (up to 12.8 μM) for 48 h. (B) Schematic representation of experimental workflow for the identification of acetyl-lysine-modified peptides regulated by SIRT1 inhibition. Lysates from HCT116 cells incubated with or without INZ (0.8 μM) were subjected to trypsin digestion and acetylation antibody-based enrichment, and followed by MS analysis. (C) Venn diagram showing the overlap of protein numbers in DMSO-treated and INZ-treated groups. (D) The number of SIRT1 up-regulated proteins and their subcellular distribution as determined by the COMPARTMENTS database. (E) Ingenuity Pathway Analysis (IPA) of the acetylated proteins regulated by SIRT1 (fold change ≥1.5, P < 0.05). The bar graphs showing the top 15 canonical pathways enriched by IPA. (F) Description of the SIRT1-regulated acetylation sites and their subcellular distribution. (G) Histogram showing the distributions of the number of acetylation sites (left) and SIRT1-regulated sites (right) per protein. Multisite-acetylated proteins (MAPs) with more than 4 acetylation sites are indicated in red pane. (H) DAVID Gene Ontology (GO) analysis of hyperacetylated proteins regulated by SIRT1 inhibition. Significantly enriched pathways (FDR <0.01) were categorized by GO different modules for visualization (BP: biological process; CC: cellular component; MF: molecular function). (I) Pie chart showing the proportion of MAPs in mitochondrial, nuclear and cytoplasmic fractions according to cellular component analysis, as well as the distribution of MAPs in mitochondria inner membrane (MIM), mitochondria outer membrane (MOM) and matrix. (J) The acetylation level of mitochondria extracted from HCT116 cells treated with 0.8 and 3.2 μM of INZ. Tom20 and Tubulin were used as markers for mitochondria and cytoplasma, respectively. (K) The expression level of SIRT1 in different cellular components extracted from HCT116 cells, respectively. Nuc, nucleus; mito, mitochondria; cyto, cytosol. (L) Western blot analysis confirming the effect of SIRT1 knockout in HCT116 cells. (M) Western blot analysis of the acetylation level of mitochondria in HCT116 cells after knockout of SIRT1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)