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. 2012 Feb 1;26(3):259–270. doi: 10.1101/gad.180406.111

Figure 5.

Figure 5.

Sirt2 promotes oxidative catabolism via Pgc1α deacetylation. (A) 3T3-L1-derived adipocytes were infected with empty nsRNA, shHif1α, shSirt1, shSirt2, or a combination thereof and assessed for oleate-induced oxygen consumption as a measure of fatty acid β-oxidation using the Seahorse Bioscience 24XF extracellular flux analyzer. The normalized OCR is shown. (*) P < 0.01 compared with control nsRNA, set at 1.0; (%) P < 0.01 compared with shHif1α; (ç) P < 0.01 compared with shSirt1. Data are mean ± SEM of values from each group. (B) 3T3-L1-derived adipocytes were infected with empty nsRNA, shPgc1α, shHif1α, or shHif1α/shPgc1α and assessed for oleate-induced oxygen consumption as a measure of fatty acid β-oxidation using the Seahorse Bioscience 24XF extracellular flux analyzer. The normalized OCR is shown. (*) P < 0.01 compared with control nsRNA, set at 1.0; (ç) P < 0.01 compared with shHif1α. Data are mean ± SEM of values from each group. (C) Lentivirus-mediated ectopic expression of Sirt2 was performed in 3T3-L1-derived adipocytes in the presence or absence of shPgc1α and assessed for oleate-induced oxygen consumption as a measure of fatty acid β-oxidation using the Seahorse Bioscience 24XF extracellular flux analyzer. The normalized OCR is shown. (*) P < 0.01 compared with control nsRNA, set at 1.0; (ç) P < 0.01 compared with Sirt2. Data are mean ± SEM of values from each group. (D, top panels) Ectopic lentiviral-mediated Sirt2 or Sirt2 H187A expression was performed in 293 cells, and lysates were immunoprecipitated with the Pgc1α antibody and probed for lysine acetylation and Pgc1α by immunoblotting. (Bottom panels) In parallel, lysates were probed for Sirt2 expression and tubulin acetylation by immunoblotting. (E, top panels) Lentiviral shRNA-mediated Sirt1 and Sirt2 knockdown was performed in 293 cells, and lysates were immunoprecipitated with the Pgc1α antibody and probed for lysine acetylation and Pgc1α by immunoblotting. (Bottom panels) In parallel, lysates were probed for Sirt1 and Sirt2 expression by immunoblotting. (F) Flag-tagged Pgc1α ectopically expressed in 293 cells was immunoprecipitated and incubated in the presence of varying amounts of purified Sirt2 protein in the presence or absence of NAD+ as indicated and probed by immunoblotting for acetylated lysine and Pgc1α. Immunoprecipitation with IgG serves as a negative control.