pad track flagsirt1 h355a  (Addgene inc)

Journal: PLoS Biology

Article Title: Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

doi: 10.1371/journal.pbio.1001603

Figure Lengend Snippet: (A) Nicotinamide (Nic) decreases SIRT1 activity in C 2 C 12 myotubes as evidenced by increased p53 acetylation and prevention of p53 deacetylation in response to resveratrol (RSV). * p <0.05 versus control; # p <0.05 versus RSV and RSV+Nic. (B) Inhibition of SIRT1 with nicotinamide (Nic) does not prevent the resveratrol (RSV)-induced increase in mitochondrial proteins. * p <0.05 versus control. (C) Suppression of SIRT1 activity with nicotinamide or with dominant-negative SIRT1 H355A results in increased acetylation of PGC-1α. SIRT1 H355A reduces PGC-1α deacetylation in response to resveratrol (RSV) treatment, while overexpression of wild-type (WT) SIRT1 results in PGC-1α deacetylation. Values are means ± SE for 6–8 experiments. * p <0.05 versus control; # p <0.05 versus other groups. (D) PGC-1α coactivator activity, measured in C 2 C 12 myotubes co-transfected with a PGC-1α GAL4 fusion product and a luciferase reporter, was increased by treatment with 20 µM resveratrol (RSV). Overexpression of wild-type (WT) SIRT1 resulted in reduced PGC-1α coactivator activity. Suppression of SIRT1 activity with dominant-negative SIRT1 H355A or knockdown of SIRT1 with SIRT1 shRNA resulted in increases in PGC-1α coactivator activity and potentiation of the effect of resveratrol on PGC-1α activity. In the experiments in which C 2 C 12 myotubes were treated with 50 µM resveratrol, PGC-1α was overexpressed in the myotubes (see and text). Values are means ± SE for 6–7 experiments. * p <0.05 versus control; # p <0.05 versus basal.

Article Snippet: For expression in skeletal muscle via electroporation, wild-type SIRT1 and dominant-negative SIRT-1 H355A constructs were purchased from Addgene (Cambridge, MA) and inserted into pCDNA3.1 (Invitrogen, Carlsbad, CA).

Techniques: Activity Assay, Control, Inhibition, Dominant Negative Mutation, Over Expression, Transfection, Luciferase, Knockdown, shRNA

Journal: PLoS Biology

Article Title: Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

doi: 10.1371/journal.pbio.1001603

Figure Lengend Snippet: (A) Suppression of SIRT1 activity with dominant-negative SIRT1 H355A in C 2 C 12 myotubes increases mitochondrial proteins. Values are means ± SE for 6–10 experiments per group. p <0.5 versus control. (B) Suppression of SIRT1 activity by expression of dominant-negative SIRT1 H355A in rat triceps muscle by electroporation resulted in increases in mitochondrial proteins. Values are means ± SE for 5–7 muscles per group. * p <0.05 versus control. (C) Knockdown of SIRT1 with a SIRT1 shRNA in C 2 C 12 myotubes resulted in increases in mitochondrial proteins. Values are means ± SE for 6–8 experiments per group. * p <0.05 versus control. (D) Overexpression of wild-type (WT) SIRT1 in C 2 C 12 myotubes resulted in decreased cytochrome c protein expression and inhibited the resveratrol (RSV)-induced increase in cytochrome c. Values are means ± SE for 6 experiments. * p <0.05 versus control. # p <0.05 versus other groups. (E) Overexpression of wild-type (WT) SIRT1 in rat triceps muscle by electroporation resulted in decreased expression of mitochondrial proteins. Values are means ± SE for 7–8 muscles per group. * p <0.05 versus empty vector.

Article Snippet: For expression in skeletal muscle via electroporation, wild-type SIRT1 and dominant-negative SIRT-1 H355A constructs were purchased from Addgene (Cambridge, MA) and inserted into pCDNA3.1 (Invitrogen, Carlsbad, CA).

Techniques: Activity Assay, Dominant Negative Mutation, Control, Expressing, Electroporation, Muscles, Knockdown, shRNA, Over Expression, Plasmid Preparation

Journal: PLoS Biology

Article Title: Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

doi: 10.1371/journal.pbio.1001603

Figure Lengend Snippet: (A) Nicotinamide (Nic) decreases SIRT1 activity in C 2 C 12 myotubes as evidenced by increased p53 acetylation and prevention of p53 deacetylation in response to resveratrol (RSV). * p <0.05 versus control; # p <0.05 versus RSV and RSV+Nic. (B) Inhibition of SIRT1 with nicotinamide (Nic) does not prevent the resveratrol (RSV)-induced increase in mitochondrial proteins. * p <0.05 versus control. (C) Suppression of SIRT1 activity with nicotinamide or with dominant-negative SIRT1 H355A results in increased acetylation of PGC-1α. SIRT1 H355A reduces PGC-1α deacetylation in response to resveratrol (RSV) treatment, while overexpression of wild-type (WT) SIRT1 results in PGC-1α deacetylation. Values are means ± SE for 6–8 experiments. * p <0.05 versus control; # p <0.05 versus other groups. (D) PGC-1α coactivator activity, measured in C 2 C 12 myotubes co-transfected with a PGC-1α GAL4 fusion product and a luciferase reporter, was increased by treatment with 20 µM resveratrol (RSV). Overexpression of wild-type (WT) SIRT1 resulted in reduced PGC-1α coactivator activity. Suppression of SIRT1 activity with dominant-negative SIRT1 H355A or knockdown of SIRT1 with SIRT1 shRNA resulted in increases in PGC-1α coactivator activity and potentiation of the effect of resveratrol on PGC-1α activity. In the experiments in which C 2 C 12 myotubes were treated with 50 µM resveratrol, PGC-1α was overexpressed in the myotubes (see and text). Values are means ± SE for 6–7 experiments. * p <0.05 versus control; # p <0.05 versus basal.

Article Snippet: For expression by adenoviral infection in C 2 C 12 myotubes, the adenoviral constructs of pAd-Track Flag-PGC-1α , pAd-Track Flag-SIRT1 , and pAd-Track Flag dominant-negative SIRT1 H355A were purchased from Addgene (Cambridge, MA).

Techniques: Activity Assay, Control, Inhibition, Dominant Negative Mutation, Over Expression, Transfection, Luciferase, Knockdown, shRNA

Journal: PLoS Biology

Article Title: Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

doi: 10.1371/journal.pbio.1001603

Figure Lengend Snippet: (A) Suppression of SIRT1 activity with dominant-negative SIRT1 H355A in C 2 C 12 myotubes increases mitochondrial proteins. Values are means ± SE for 6–10 experiments per group. p <0.5 versus control. (B) Suppression of SIRT1 activity by expression of dominant-negative SIRT1 H355A in rat triceps muscle by electroporation resulted in increases in mitochondrial proteins. Values are means ± SE for 5–7 muscles per group. * p <0.05 versus control. (C) Knockdown of SIRT1 with a SIRT1 shRNA in C 2 C 12 myotubes resulted in increases in mitochondrial proteins. Values are means ± SE for 6–8 experiments per group. * p <0.05 versus control. (D) Overexpression of wild-type (WT) SIRT1 in C 2 C 12 myotubes resulted in decreased cytochrome c protein expression and inhibited the resveratrol (RSV)-induced increase in cytochrome c. Values are means ± SE for 6 experiments. * p <0.05 versus control. # p <0.05 versus other groups. (E) Overexpression of wild-type (WT) SIRT1 in rat triceps muscle by electroporation resulted in decreased expression of mitochondrial proteins. Values are means ± SE for 7–8 muscles per group. * p <0.05 versus empty vector.

Article Snippet: For expression by adenoviral infection in C 2 C 12 myotubes, the adenoviral constructs of pAd-Track Flag-PGC-1α , pAd-Track Flag-SIRT1 , and pAd-Track Flag dominant-negative SIRT1 H355A were purchased from Addgene (Cambridge, MA).

Techniques: Activity Assay, Dominant Negative Mutation, Control, Expressing, Electroporation, Muscles, Knockdown, shRNA, Over Expression, Plasmid Preparation