Animal Experiments and Treatments Price et al., 2012 Mouchiroud L.

Houtkooper R.H.

Moullan N.

Katsyuba E.

Ryu D.

Cantó C.

Mottis A.

Jo Y.S.

Viswanathan M.

Schoonjans K.

et al. The NAD(+)/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Price et al., 2012 Mouchiroud L.

Houtkooper R.H.

Moullan N.

Katsyuba E.

Ryu D.

Cantó C.

Mottis A.

Jo Y.S.

Viswanathan M.

Schoonjans K.

et al. The NAD(+)/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Minamishima et al., 2008 Hubbard B.P.

Gomes A.P.

Dai H.

Li J.

Case A.W.

Considine T.

Riera T.V.

Lee J.E.

E S.Y.

Lamming D.W.

Pentelute B.L.

et al. Evidence for a common mechanism of SIRT1 regulation by allosteric activators. C57BL/6J mice of 6, 22, and 30 months of age were obtained from the National Institutes of Aging mouse aging colony. Additionally 22-month-old caloric restricted mice were also obtained from the National Institutes of Aging mouse aging colony. Mice were acclimated for at least one-week prior to sacrifice. 6 and 22-month-old mice were given interperitoneal (IP) injections of 500 mg NMN/kg body weight per day or the equivalent volume of PBS for 7 consecutive days at 6:00 pm and 8:00 am on day 8 and sacrificed 4 hr after last injection. Whole body adult-inducible SIRT1 knockout mice (SIRT1 iKO) () were treated with tamoxifen for 5 weeks and the efficiency of deletion in DNA from tail samples was determined by PCR. Animals were then maintained on regular diet for 2-6 months. For fasting experiments, mice were fasted for 16 hr prior to sacrifice. For NMN experiments both WT and SIRT1 iKO mice were given IP injections of 500 mg NMN/kg body weight per day or the equivalent volume of PBS for 7 consecutive days at 6:00 pm and 8:00 am on day 8 and sacrificed 4 hr after last injection. Whole body SIRT1 overexpressor (SIRT1-Tg) mice () of 6 months of age were given IP injections of 300 mg DMOG/kg body weight per day or the equivalent volume of PBS for 5 consecutive days, after which they were sacrificed. Whole body adult-inducible EglN1 knockout mice () were treated with IP injection of tamoxifen for 3 days after which they were allowed to rest for an additional 3 days. The mice were then given IP injections of 500 mg NMN/kg body weight per day or the equivalent volume of PBS for 7 consecutive days at 6:00 pm and 8:00 am on day 8 and sacrificed 4 hr after last injection. All animal studies followed the guidelines of and were approved by the Harvard Institutional Animal Care and Use Committee.

Overexpression of NMNAT1 In Vivo The NMNAT1 plasmid was a kind gift From Professor Toshiyuki Araki (National Center of Neurology and Psychiatry, Japan) and was generated by cloning mouse NMNAT1 cDNA with a C-terminal 6xHis-Tag into the BamH1 site of a pIRES-eGFP plasmid (Clonetech). The plasmid was purified using endotoxin-free Maxi-Prep Kit (QIAGEN) and resuspended in sterile 0.9% saline. Mice were anaesthetized, their hind limbs shaved and wiped with ethanol. Hyaluronidase (15 units in 50 μl saline) was injected into the tibialis cranialis muscle transcutaneously along the length of the muscle using an insulin syringe. Following a 1.5 hr waiting period, plasmid (50 μg in 50 μl saline) was injected along the complete length of one tibialis muscle. This was followed by eight pulses of 100 V/cm and 20 msec at a frequency of 1 Hz via tweezer electrodes attached to an ECM-830 electroporator (BTX). For each mouse, the contralateral muscle received an equal volume saline and underwent the electroporation protocol, to act as an internal control. Mice were sacrificed 7 days following electroporation and both tibialis muscles were collected for subsequent analyses. Animal experiments followed the guidelines and were approved by the Garvan Institute, Australia.

Ex Vivo Incubation of Soleus with Insulin and Determination of Insulin-Stimulated Glucose Uptake Wu et al., 2009 Singer T.P. Determination of the activity of succinate, NADH, choline, and alpha-glycerophosphate dehydrogenases. Ex-vivo incubation of soleus with insulin was performed as described before () with slight modifications. Briefly, two strips of soleus muscle were isolated and incubated in Krebs Henseleit buffer, pH 7.4, containing 20 mM HEPES and 2% bovine serum albumin (KHB) at 37°C for 20 min. After pre-incubation in KHB, muscles were incubated in KHB supplemented with 5.5 mM glucose, and 2 mM pyruvate for another 30 min (control), or in the same buffer but containing human recombinant insulin (100 μU/mL) for 30 min. For insulin stimulated glucose uptake, the samples were incubated with 2-deoxyglucose (2-DG), an analog of glucose which is known to be transported by the same glucose carrier system as glucose and phosphorylated to 2-deoxyglucose-6-phosphate but not metabolized further. Samples were then washed with KHB, immediately frozen in liquid nitrogen, and stored at −80°C. 2-DG uptake was measured using a commercially available kit (BioVision) according to the manufacturer’s instructions.

Adenovirus Cloning and Production TFAM and control (GFP) adenoviruses were constructed by cloning the inserts into the pAd/CMV/V5-DEST Gateway Vector to generate adenoviral expression vectors. The adenoviral expression vectors were digested with PacI for 30 min. The linearized adenoviral expression vectors were then transfected using X-tremeGENE HP into 293 cells to produce adenoviruses. The media was changed the next day and the transfected 293 cells were maintained in culture to monitor for cytopathic effect. The adenoviruses were harvested when 80% of cytopathic effect was observed and used as crude adenoviral lysates.

C2C12 Cell Culture, Treatments, and Adenoviral Infections Gerhart-Hines et al., 2007 Gerhart-Hines Z.

Rodgers J.T.

Bare O.

Lerin C.

Kim S.H.

Mostoslavsky R.

Alt F.W.

Wu Z.

Puigserver P. Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha. C2C12 cell line (ATCC) was cultured in low glucose Dulbecco’s modified eagle medium (DMEM) (Invitrogen) supplemented with 10% FBS (Invitrogen) and a mix of antibiotic and antimycotic (Invitrogen). To inhibit SIRT1, cells were treated the vehicle (0.001% DMSO) or 10 μM EX-527 (Tocris) for 24 hr. C2C12 myoblasts were infected with an empty or SIRT1 adenovirus as described before () and the media was replaced with fresh DMEM for an additional 48 hr.

Generation of Primary Myoblasts from CreERT2 SIRT1 Flox/Flox Mice, Induction of SIRT1 Exon4 Excision, Adenoviral Infections, and Treatments Price et al., 2012 Mouchiroud L.

Houtkooper R.H.

Moullan N.

Katsyuba E.

Ryu D.

Cantó C.

Mottis A.

Jo Y.S.

Viswanathan M.

Schoonjans K.

et al. The NAD(+)/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Hubbard et al., 2013 Hu C.J.

Sataur A.

Wang L.

Chen H.

Simon M.C. The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha. Hubbard et al., 2013 Hu C.J.

Sataur A.

Wang L.

Chen H.

Simon M.C. The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha. Primary myoblasts cells were isolated from CreERT2 SIRT1 flox/flox () as previously described (). To induce the excision of exon 4 of SIRT1 the cells were treated with 250 nM 4-hydroxytamoxifen (Sigma-Aldrich), or vehicle (0.001% ethanol) as previously described () for the time points indicated. For testing the effect of AMPK activation on the regulation of mitochondrial-encoded and nuclear encoded genes, CreERT2 SIRT1 flox/flox cells were infected with an AMPKα2-DN adenovirus (Eton Bioscience) for 48 hr during which 250 nM 4-hydroxytamoxifen was added to promote SIRT1 exon 4 excision. For TFAM addback experiments, SIRT1 excision was induced with 250 nM 4-hydroxytamoxifen for either 24 or 48 hr after which the cells were infected with control or TFAM adenovirus for an additional 24 hr. To address the role of SIRT1 on NMNAT1’s effects, CreERT2 SIRT1 flox/flox myoblasts were infected with adenovirus expressing empty or NMNAT1 (Applied Biological Materials) and simultaneously SIRT1 excision was induced with with 250 nM 4-hydroxytamoxifen for 24 hr. To evaluate the effects of SIRT1 on HIF-1α hydroxylation, CreERT2 SIRT1 flox/flox cells were treated for 6 hr with 20 μM MG132 (Cayman) after SIRT1 was excised for 24 hr. Un-induced CreERT2 SIRT1 flox/flox cells were used as WT primary myoblasts for subsequent experiments and genetic manipulations.

Generation of RhoO Cells CreERT2 SIRT1 flox/flox primary myoblasts were mantained in normal growth media (F-10 nutrient mix supplemented with 2.5 ng/mL bFGF, 10 ng/mL EGF, 1 μg/mL insulin, 0.5 mg/mL fetuin. 0.4 μg/mL dexamethasone, antibiotic and antimycotic mix and 20% FBS) supplemented with 4 g/L glucose and 2 mM pyruvate, 50 ng/ml ethidium bromide (Alfa aesar) and 100 μg/mL uridine (Sigma-Aldrich) for 4 weeks to deplete mitochondrial DNA (rho0 cells). Parental cells were cultured in similar media without ethidium bromide and uridine for the same period of time and used as control cells. After 4 weeks of treatment the amount of mtDNA was checked by qPCR and mitochondrial-encoded subunits tested by Western blot to validate the cell model.

Generation, Culture, and Treatments of Primary Myoblasts from PGC-1α/β KO and Muscle-Specific PGC-1α Null Mice Zechner et al., 2010 Yoshino J.

Mills K.F.

Yoon M.J.

Imai S. Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Handschin et al., 2007 Handschin C.

Choi C.S.

Chin S.

Kim S.

Kawamori D.

Kurpad A.J.

Neubauer N.

Hu J.

Mootha V.K.

Kim Y.B.

et al. Abnormal glucose homeostasis in skeletal muscle-specific PGC-1alpha knockout mice reveals skeletal muscle-pancreatic beta cell crosstalk. Hubbard et al., 2013 Hu C.J.

Sataur A.

Wang L.

Chen H.

Simon M.C. The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha. Gerhart-Hines et al., 2007 Gerhart-Hines Z.

Rodgers J.T.

Bare O.

Lerin C.

Kim S.H.

Mostoslavsky R.

Alt F.W.

Wu Z.

Puigserver P. Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha. Primary myoblasts were isolated from PGC-1α/β KO () and muscle specific PGC-1α null mice () as previously described (). WT, PGC-1α/β KO and PGC-1α null primary myoblasts were plated and allowed to differentiate into myotubes by replacing the media with low glucose DMEM supplemented with 2% horse serum (Sigma-Aldrich) for 5 days. After the differentiation the cells were infected with empty vector or flag-SIRT1 adenovirus as described before (). Media was replaced with fresh DMEM supplemented with 2% horse serum (Sigma-Aldrich) for an additional 48 hr and, after that the cells were harvested for the different assays as described. To investigate the role of HIF-1α in SIRT1-mediated induction of mitochondrial-encoded genes, PGC-1α/β KO and PGC-1α null primary myotubes were treated for 12 hr with vehicle (0.001% DMSO) or 1mM DMOG (Sigma-Aldrich), 24 hr after infection with empty vector or flag-SIRT1 adenovirus. To study how nuclear energetics influence PGC-1α/β independent pathways, PGC-1α/β KO primary myotubes were infected with empty vector or NMNAT1 adenovirus (Applied Biological Materials) for 24 hr.

NMNAT1, NMNAT2, NMNAT3, VHL, c-Myc, and HF1α Gene Silencing in Primary Myoblasts shNMNAT1#1 (TRCN0000035471; Open Biosystems), shNMNAT1#2 (TRCN0000035473; Open Biosystems), shNMNAT2#1 (TRCN0000111479; Open Biosystems), shNMNAT2#2 (TRCN0000111476; Open Biosystems), shNMNAT3#1 (TRCN0000035402; Open Biosystems), shNMNAT3#2 (TRCN0000035400; Open Biosystems), shMyc#1 (TRCN0000042517; Open Biosystems) ShMyc#2 (TRCN0000054885; Open Biosystems), shHIF-1α (TRCN0000054450; Open Biosystems), shVHL#1 (TRCN0000009736; Open Biosystems), shVHL#2 (TRCN0000009734; Open Biosystems) and control shGFP lentivirus were produced by co-transfection of 293T cells with plasmids encoding psPAX2 (Addgene plasmid 12260), and pMD2.G (Addgene plasmid 12259) using X-tremeGENE HP (Roche) in accordance with the manufacturer’s protocol. Media was changed 24 hr post-transfection and the virus harvested after 48 hr, filtered and used to infect CreERT2 SIRT1 flox/flox primary myoblasts in the presence of 5 μg/mL polybrene (Sigma-Aldrich) via spin infection (2500 rpm, 30 min). Selection of resistant colonies was initiated 24 hr later using 2 μg/mL puromycin (Invivogen). To evaluate the requirement of NMNAT1 for NMN’s effects, NMNAT1 knockdown, primary myoblasts were treated with vehicle (PBS) or 500 μM NMN (Sigma-Aldrich) for 24 hr.

ARNT and c-Myc Gene Silencing in C2C12 Cells shMyc#1 (TRCN0000042517; Open Biosystems), shMyc#2 (TRCN0000054885; Open Biosystems), shARNT#1 and shANRT#2 (TRCN0000079930 and TRCN0000079931, respectively; Open Biosystems) and control shGFP lentivirus were produced and C2C12 cells were transduced as described above.

HIF-1α and HIF-2α DPA in C2C12 Cells pBabe empty (Addgene plasmid 1764), HIF-1α DPA (Addgene plasmid 19005), and HIF-2α DPA (Addgene plasmid 19006) retrovirus were produced by co-transfection of 293T cells with plasmids encoding gagpol (Addgene plasmid 14887) and vsvg (Addgene plasmid 8454) using X-tremeGENE HP (Roche) in accordance with the manufacturer’s protocol. Media was changed 24 hr post-transfection and the virus harvested after 48 hr, filtered and used to infect C2C12 cells in the presence of 5 μg/mL polybrene (Sigma-Aldrich) via spin infection (2500 rpm, 30 min). Selection of resistant colonies was initiated 24 hr later using 2 μg/mL puromycin (Invivogen).

c-Myc Overexpression in C2C12 Cells and Primary Myoblasts pMXsc-Myc (Addgene plasmid 13375) and empty retrovirus were produced as described above and C2C12 cells or CreERT2 SIRT1 flox/flox primary myoblasts were transduced as described above.

HIF-1α Overexpression and Mutagenesis Mutagenesis of HIF-1α (Addgene plasmid 19365) was performed with QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) and using the following primers: K709Q sense: 5′-cctgaggaagaactaaatccacagatactagctttgcag-3′, antisense: 3′-ggactccttcttgatttaggtgtctatgatcgaaacgtc-5′; K709R sense: 5′-ctgaggaagaactaaatccaaggatactagctttgcagaatgc-3′, antisense: 3′-gactccttcttgatttaggttcctatgatcgaaacgtcttacg-5′; K674Q sense: 5′-cctcaccaaacagagcaggacagggagtcatagaacagaca-3′, antisense: 3′-ggagtggtttgtctcgtcctgtccctcagtatcttgtctgt-5′ HIF-1α WT and mutant overexpressing cells were made using pBabe puromycin resistant vectors by retroviral transduction as described above.

Mitochondrial Membrane Potential, Reactive Oxygen Species, and Mitochondrial Mass Measurements Bell et al., 2011 Bell E.L.

Emerling B.M.

Ricoult S.J.

Guarente L. SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production. Gomes et al., 2012 Gomes A.P.

Duarte F.V.

Nunes P.

Hubbard B.P.

Teodoro J.S.

Varela A.T.

Jones J.G.

Sinclair D.A.

Palmeira C.M.

Rolo A.P. Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis. Mitochondrial membrane potential was evaluated by fluorescence of the potential dependent TMRM probe. Briefly, cells were incubated with 100 nM TMRM for 15 min in the dark, after which the media was replaced and the fluorescence was measure by flow cytometry. Reactive oxygen species and mitochondrial mass were also evaluated by flow cytometry using the fluorescent probes DHE and NAO respectively as described before ().

Succinate Dehydrogenase Activity 2 consumption using phenazine metasulphate (PMS) as an artificial electron acceptor, as previously described ( Singer, 1974 Santos J.H.

Meyer J.N.

Mandavilli B.S.

Van Houten B. Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Succinate dehydrogenase activity was polarographically determined based on the Oconsumption using phenazine metasulphate (PMS) as an artificial electron acceptor, as previously described (). The reaction was carried out at 25°C in 1.3 ml of standard respiratory medium (as in mitochondrial respiration) supplemented with 5 mM succinate, 2 μM rotenone, 0.1 μg antimycin A, 1 mM KCN and 0.3 mg Triton X- 100. After the addition of the sample, the reaction was initiated with 1 mM PMS.

Cytochrome c Oxidase Activity 2 consumption upon cytochrome c oxidation, as previously described ( Brautigan et al., 1978 Ahuja P.

Zhao P.

Angelis E.

Ruan H.

Korge P.

Olson A.

Wang Y.

Jin E.S.

Jeffrey F.M.

Portman M.

Maclellan W.R. Myc controls transcriptional regulation of cardiac metabolism and mitochondrial biogenesis in response to pathological stress in mice. Cytochrome c oxidase activity was polarographically determined based on the Oconsumption upon cytochrome c oxidation, as previously described (). The reaction was carried out at 25°C in 1.3 ml of standard respiratory medium (as in mitochondrial respiration) supplemented with 2 μM rotenone, 10 μM oxidized cytochrome c, 0.3 mg Triton X-100. Following addition of the sample, the reaction was initiated by adding 5 mM ascorbate plus 0.25 mM tetramethylphenylene-diamine (TMPD). Determination of COX activity by spectrophotometry in protein extracts from the skeletal muscle was performed using a commercially available kit (Sigma-Aldrich) according to the manufacturer’s instructions.

Electron Microscopy Skeletal muscle from mice were fixed in 2.5% glutaraldehyde and 2.5% paraformaldehyde in cacodylate buffer (Electron Microscopy Sciences) then were removed, put directly into fixative, then were embedded and photographed with an electron microscope (Tecnai G2 Spirit BioTWIN). Mitochondrial area was quantified with Image J software.

Analysis of mtDNA Integrity Santos et al., 2006 Rolo A.P.

Palmeira C.M.

Wallace K.B. Mitochondrially mediated synergistic cell killing by bile acids. Total DNA was extracted with DNeasy blood and tissue kit (QIAGEN). Integrity of mtDNA was assessed using the long range PCR mediated detection method as described previously (), using the following primer sequences: Fwd: GCCAGCCTGACCCATAGCCATAATAT; Rev: GAGAGATTTTATGGGTGTAATGCGG.

Gene Expression and mtDNA Analysis -ΔCT) using 18S as an internal control. For mtDNA analysis, total DNA was extracted with DNeasy blood and tissue kit (QIAGEN) according to the manufacturer’s instructions. mtDNA was amplified using primers specific for the mitochondrial cytochrome c oxidase subunit 2 (COX2) gene and normalized to genomic DNA by amplification of the ribosomal protein s18 (rps18) nuclear gene. Primers were designed using the IDT software (IDT) or obtained from primer bank ( RNA from skeletal muscle tissue was extracted using the RNeasy for fibrous tissue mini kit (QIAGEN) according to the instructions. RNA from C2C12 cells and primary myoblasts were extracted with RNeasy mini kit (QIAGEN) also according to the instructions. RNA from brain, liver, heart and white adipose tissue was extracted using Trizol (Invitrogen) according to the manufacture’s instructions. RNA samples were quantified using the NanoDrop 1000 spectrophotometer (Thermo Scientific). cDNA was synthesized with the iSCRIP cDNA synthesis kit (BioRad) using 600 ng of RNA. Quantitative RT-PCR reactions were performed using 1 μM of primers and LightCycler® 480 SYBR Green Master (Roche) on a LightCycler® 480 detection system (Roche). Calculations were performed by a comparative method (2) using 18S as an internal control. For mtDNA analysis, total DNA was extracted with DNeasy blood and tissue kit (QIAGEN) according to the manufacturer’s instructions. mtDNA was amplified using primers specific for the mitochondrial cytochrome c oxidase subunit 2 (COX2) gene and normalized to genomic DNA by amplification of the ribosomal protein s18 (rps18) nuclear gene. Primers were designed using the IDT software (IDT) or obtained from primer bank ( http://pga.mgh.harvard.edu/primerbank/citation.html ) and the primer sequences can be found in the Table S1.

Chromatin Immunoprecipitation Ahuja et al., 2010 Zhong L.

D’Urso A.

Toiber D.

Sebastian C.

Henry R.E.

Vadysirisack D.D.

Guimaraes A.

Marinelli B.

Wikstrom J.D.

Nir T.

et al. The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Hu et al., 2007 Emaus R.K.

Grunwald R.

Lemasters J.J. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Chromatin immunoprecipitation was performed using a commercial available kit (Millipore) according to the manufacturer’s instructions and using anti-HIF1α (Cayman) and anti-c-Myc (Abcam) antibodies and utilizing the following primer sequences: TFAM promoter Fw: TTGGCTGGCTAAGCTCATCT, Rv: AAGGCTGAGAAGCGATAGCA (); LDHA Fw: ATCGATGCATTTGGGCTC, Rv: CAACCCGACATGCTCCTCA ().

Coimmunoprecipitation Proteins from CreERT2 SIRT1 flox/flox primary myoblasts were crosslinked using 1 mM DSP (Pierce) after which the cells were lysed in a low-stringency IP buffer (0.05% NP-40, 50 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl, pH 7.4) supplemented with protease Inhibitor cocktail (Roche) and 25 U/ml endonuclease (Pierce). Endogenous HIF-1α protein was imunoprecipitated using anti-HIF-1α antibody (Cayman) coated A/G magnetic beads (Pierce). Anti-rabbit IgG antibody (Milipore) coated A/G magnetic beads were used as a control. Immunoprecipitated material was washed ten times for 25 min each in low stringency lysis buffer, after which it was eluted in SDS-PAGE buffer supplemented with 50 mM DTT and boiled for 10 min. Imunoprecipitated proteins and input were run on SDS-PAGE under reducing conditions. The separated proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane (Perkin-Elmer). Proteins of interest were revealed with anti-HIF-1α (Cayman) and anti-c-Myc (Abcam) antibodies overnight at 4°C. The immunostaining was detected using Clean-Blot IP detection reagent (Pierce) according to the manufacturer’s instructions. Bands were revealed using Amersham ECL detection system (GE Healthcare).

Immunoblot Protein extracts from tissue or C2C12 cells were obtained by lysis in ice-cold lysis buffer (150 mM NaCl,10 mM Tris HCl (pH 7.4),1 mM EDTA,1 mM EGTA, 1% Triton X-100, 0.5% NP-40) supplemented with a cocktail of protease and phosphatase inhibitors (Roche). Protein content was determined by the Bradford protein assay (Biorad), and 50 μg proteins were run on SDS-PAGE under reducing conditions. The separated proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane (Perkin-Elmer). Proteins of interest were revealed with specific antibodies: anti-TFAM (Aviva biosciences), anti-COX2, anti-COX4 (Mitosciences), anti-SIRT1, anti-β-tubulin, anti-Flag (Sigma-Aldrich), anti-HIF1α (Cayman), anti-HA (Covance), anti-HIF-2α, anti-NMNAT1 (Novus Biologicals), Anti-NMNAT2, anti-c-Myc, anti-MuRF1, anti-Atrogin1 (Abcam), Anti-NMNAT3 (Santa Cruz Biotecnologies), anti-HIF-1α-OH, anti-VHL, anti-pAMPK (Thr172), anti-AMPK, anti-pACC (Ser79), anti-ACC, anti-pATK (Ser473), anti-AKT (Cell Signaling), anti-pIRS1 (Tyr608), anti-IRS1 (Milipore), anti-MyHCIIa and anti-MyHCIIb (Developmental Studies Hybridoma bank, University of Iowa) overnight at 4°C. The immunostaining was detected using horseradish peroxidase–conjugated anti-rabbit, anti-mouse or anti-goat immunoglobulin for 1 hr at room temperature. Bands were revealed using Amersham ECL detection system (GE Healthcare).

TFAM Promoter Activity and TFAM Promoter Mutagenesis TFAM promoter activity was evaluated using a TFAM promoter-luciferase plasmid. A fragment of the mouse TFAM promoter (1.4kb upstream of the coding sequence) was cloned into a pGL4.15 vector (Promega). c-Myc binding site was mutated using the QuickChange lightning kit (Stratagene) according to the manufacturer’s instructions and using the following primer sequences: sense: 5′-ggtttggctggctaagctcatctagtcaaaaaaatctgcaaagtgggaaacaatattcag-3′ antisense: 5′-ctgaatattgtttcccactttgcagatttttttgactagatgagcttagccagccaaacc-3′. Primary myoblasts were transfected with either with TFAM-luciferase full length promoter or c-myc binding site mutant using X-tremeGENE HP (Roche) in accordance with the manufacturer’s protocol. For SIRT1 and PGC-1α overexpression experiments cells were infected with the respective adenovirus or empty vector 24 hr after transfection with the luciferase plasmid. For c-Myc overexpression or c-Myc knockdown primary cells were produced as described above and transfected with either with TFAM-luciferase full length promoter or c-myc binding site mutant using X-tremeGENE HP (Roche) in accordance with the manufacturer’s protocol. To access TFAM promoter in SIRT1 iKO, CreERT2 SIRT1flox/flox primary myoblasts were transfected with TFAM-luciferase full length promoter or c-myc binding site mutant using X-tremeGENE HP (Roche) in accordance with the manufacturer’s protocol and 24 hr after treated either with 250 nM 4-hydroxytamoxifen (Sigma-Aldrich), or vehicle (0.001% ethanol) for additional 24 hr. To assess TFAM promoter in response to HIF-1α stabilization, primary myoblasts were transfected with TFAM-luciferase full length promoter or c-myc binding site mutant using X-tremeGENE HP (Roche) in accordance with the manufacturer’s protocol and 24 hr after treated either with 1mM DMOG (Sigma-Aldrich), or vehicle (0.001% DMSO) for an additional 12 hr. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) with Renilla as the reference.