Drosophila strains and culture conditions

WT, dSesnXP4 (UAS-dSesn) and dSesn8A11 (dSesn−/−) were previously produced in Exelixis w1118 background14. Mef2-Gal4 (#27390), MHC-GS-Gal4 (#43641), ap-Gal4 (#3041), UAS-dAKT (#8191), UAS-dAKTRNAi (#33615), UAS-dRictorRNAi (#36699), UAS-dSin1RNAi (#32371), UAS-4E-BPRNAi (#36667), UAS-dTSC2RNAi (#34737), UAS-dPGC1RNAi (#33915), and fly lines with an attP landing platform were obtained from the Bloomington Drosophila Stock Center (BDSC). UAS-dSesnRNAi-A (#38481) and UAS-dSesnRNAi-B (#104365) were obtained from the Vienna Drosophila RNAi Center (VDRC). UAS-dPGC1 is a kind gift from Dr. David Walker (UCLA), and UAS-myr-dAKT (constitutive active AKT) is from Dr. Michelle Bland (University of Virginia). dSesn cDNA14 of wild-type (dSesnWT) and C86S (dSesnCS)-, D424A (dSesnDA)- or D423A/D424A (dSesnDDAA)-mutated forms were attached to an N-terminal 3 × -FLAG tag, and cloned into a pUAST-attB vector. C86, D423 and D424 in dSesn correspond to C125, D406 and D407 in Sesn2. The constructs were microinjected into embryos of the attP (#24486 from BDSC) strain, which has a PhiC31 integrase insertion on the X chromosome and an attP landing platform on the second chromosome. The transgene insertion was identified by presence of the mini-white marker. The flies were cultured on standard cornmeal-agar medium (for strain maintenance and breeding) or 10% sugar-yeast medium (for post-development husbandry, exercise training and lifespan assay) with humidity (70%), temperature (25oC) and light (12/12 h light/dark cycle) control.

Exercise training in Drosophila

Cohorts of at least 680 flies were collected under light CO 2 anesthesia within 2 h of adult eclosion and separated into vials containing 20 flies. Flies were then separated into two groups of more than 340 flies: exercised and unexercised groups. Both unexercised and exercised groups of flies were placed on the exercise training device at the same time to control for exercise-independent environmental factors. Every 15 s, the exercise device drops the vials of flies to induce an innate negative geotaxis response in a repetitive manner. Although exercised flies can run to the top of the vial, unexercised flies were prevented from running by a foam stopper placed low in the vial. Daily exercise time was gradually increased to generate a ramped program that can improve mobility in flies. For all experiments in this study, males were used for all analyses as they are more responsive to exercise training compared to females.

The exercise training was performed at the same time of day each day shortly after lights-on and did not exceed more than 3 h. Sleep is disturbed during the period of training, but the chosen period typically consists of the lowest amount of sleep to minimize the sleep disruption. Any effects seen from the minimal disruption will also be found in the unexercised controls, because they are also placed on the machine at the same time; therefore, sleep deprivation likely does not account for any of the differences seen. More complete description of the exercise protocol, as well as detailed protocols for other analyses such as runspan and flight performance assays, can be found in our recent publication67. All biochemical analyses, including western blot and quantitative RT-PCR were performed on flies after more than 24 h of rest after a final exercise bout. Therefore, the observed molecular changes are not from acute effects but from chronic and long-term effects of exercise.

Running endurance analysis in Drosophila

Eight vials of flies from each cohort were subjected to the runspan analysis at two time points: once on day 5 and once on day 25 of adulthood. For each session, the flies were placed on the Power Tower exercise machine27 and made to climb until they no longer responded to the negative geotaxis stimulus. Monitored at 15 min intervals, a vial of flies was visually determined to be fatigued when five or fewer flies could climb higher than 1 cm after three consecutive drops. Runspans used a minimum of 8 vials containing 20 flies each. Each vial was plotted as a single datum. Runspan graphs with fewer data points indicate that two or more vials were scored as fatigued at the same time. Each experiment was performed in duplicate or triplicate, and runspans were scored blindly when possible. The time from the start of the assay to the time of fatigue was recorded for each vial, and the data analyzed using log-rank analysis in GraphPad Prism (San Diego, CA, USA).

Flight performance assays in Drosophila

Duplicate or triplicate cohorts of at least 120 flies were aged and/or exercise trained in narrow vials housing groups of 20 age-matched siblings. Acrylic sheeting with paintable adhesive was placed in the flight tube, and fly cohorts were ejected into the apparatus to record flight performance and subsequent landing height after release. Fly cohorts were introduced to the flight tester one vial at a time using a gravity-dependent drop tube in order to reduce variability. After a full cohort of flies was captured on the adhesive, the sheeting was removed to a white surface in order to digitally record the landing height of each fly. Flies with damaged wings were censored from final analysis to control for mechanical stress not related to training performance. Images were analyzed using ImageJ. Landing height was averaged and compared in Prism using a two-tailed student t test.

Controls for genetic and environmental factors in Drosophila

For dSesn knockout experiments, dSesn8A11/8A11 flies were compared to Exelixis w1118 flies, from which the mutant allele was generated14. For dSesn RNAi experiments, adult progeny was collected from the same vial over a 72-h time period, and randomly split into control (OFF) and experimental (ON) groups. ON group received 100 μM mifepristone (Cayman Chemical, Ann Arbor, MI), which activates gene switch (GS) driver, while OFF group received same trace amount of vehicle solution (70% ethanol) until the experimental endpoint. For dSesn overexpression experiments, the attP (#24486 from BDSC) strain, which was used for generating UAS-dSesnWT and mutant dSesn strains, were instead crossed to Mef2-Gal4 and used as a genetic background control. These flies were used for both exercise and lifespan assays.

Exercise-independent environmental factors were controlled for by exposing unexercised flies to the same environment in all respects except that unexercised flies were prevented from running as described above.

Mouse strains and rearing conditions

All mice used in this study are in the C57BL/6 background. Sesn1−/− mice68 and Sesn2−/−/Sesn3−/− mice25, both in the C57BL/6 background, were interbred to produce TKO mice. As formerly reported69, the TKO mice showed reduced viability and semi-sterility, but considerable number of mice survived up to adulthood without any gross developmental abnormalities. Mice were maintained in filter-topped cages and were given free access to autoclaved regular chow diet (LFD; 5L0D, Lab Diet) or HFD (S3282, Bio-Serv, when indicated) and water at the University of Michigan according to National Institutes of Health (NIH) and institutional guidelines. We complied with all relevant ethical regulations for animal testing and research. All experiments were approved by the University of Michigan Institutional Animal Care & Use Committee (Protocol numbers: PRO00007710, PRO00006772, PRO00006689, PRO00006206, and PRO00005712).

Voluntary wheel running

Voluntary running experiments were performed at the University of Michigan Frankel Cardiovascular Center – Physiology Phenotyping Core, according to their established protocols. In brief, running wheels (Comfort Wheel; Central Garden and Pet) were introduced to allow mice to voluntarily exercise. 6-month-old WT (n = 4) and Sesn1−/− (n = 5) male mice (Fig. 3), 1-year old WT (n = 3; Sesn1+/− littermate control) and Sesn1−/− (n = 4) male mice (Supplementary Fig. 2), and 5-month-old WT (n = 5) and Sesn1−/−/Sesn2−/−/Sesn3−/− (TKO, n = 4) mice (Fig. 4) were subjected to voluntary wheel running. Running wheel activity was monitored daily through cyclocomputer (Cateye) for 3 weeks. After the period, mice were kept on the wheels for 4–6 additional weeks until additional metabolic analyses including glucose tolerance tests, respirometry and insulin response studies were completed.

Glucose tolerance test and insulin response studies

For glucose tolerance tests, mice were starved for 6 h, and blood was drawn from a tail nick at the indicated time points after i.p. injection of glucose (1 g kg−1 body weight), and blood glucose was instantly measured with a one-touch ultra glucose meter (Lifescan, Inc.). For an acute insulin response study, mice were kept under surgical-plane anesthesia using isoflurane, and skeletal muscle was harvested from each leg before and after injection of insulin (0.8 U kg−1 body weight). The tissues were snap frozen and used for subsequent analyses such as immunoblotting.

Metabolic cage

Metabolic cage, body composition, and exercise respirometry experiments were performed at the Michigan Mouse Metabolic Phenotyping Center Core, according to their established protocols. In brief, oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), spontaneous movements, and food intake were measured using the Comprehensive Laboratory Monitoring System (CLAMS, Columbus Instruments). After measuring body weight, each mouse was placed into the sealed chambers (7.9″ × 4″ × 5″) individually. The study was carried out continuously for 96 h, in an environmental room set at 20–23 °C with 12–12 h (6:00PM–6:00AM) dark-light cycles. During this time, food and water were provided to the animals through the feeding and drinking devices equipped inside the chamber. The amount of food consumed by each animal was monitored through a precision balance installed under the chamber. A standard gas (20.5% O 2 and 0.5% CO 2 in N 2 ) was used to calibrate the system before each experiment. VO 2 and VCO 2 samplings were done sequentially for 5 s in a 10 min interval. Spontaneous activity was recorded every second in X and Z dimensions. The air flow rate was adjusted to keep the oxygen differential around 0.3% at resting conditions. RER was calculated as VCO 2 ∙VO 2 −1.

Body composition

Body weight, fat mass, and lean body mass were measured using an NMR analyzer (Minispec LF90II, Bruker Optics), maintained according to the manufacturer’s recommendation. Conscious mice were put into the measuring tube with minimal restraint, and the individual measurements took less than 2 min.

Exercise respirometry

VO 2 and VCO 2 were measured using the CLAMS instrument described above. Before the study, the mice were each placed into the treadmill chambers to acclimate them to the treadmill environment. For two days prior to the study, the mice were individually put into the same treadmill for 30 min each day. Mice were weighed prior to the running test. They were then individually placed into the sealed treadmill chambers (305 × 51 × 44 mm3). The slope of the treadmill was set at 25° to the horizontal. The measurements were only carried out between 9:00AM and 3:00PM on each day. During this time, the animals were run on the treadmills one at a time and the treadmill was wiped clean between each test. As described above, the CLAMS system was routinely calibrated before the experiment using the standard gas. VO 2 and VCO 2 in each chamber were sampled continuously every 5 s. The air flow rate through the chambers was set at 0.50 LPM. RER was calculated as VCO 2 ∙VO 2 −1. Total glucose oxidation and fatty acid oxidation are calculated, respectively, based on the values of VO 2 and VCO 2 using equations 1.69∙VO 2 - 1.69∙VCO 2 and 4.57∙VCO 2 - 3.23∙VO 2 , respectively. The mice were all ran under the same standard treadmill schedule, which was: 30 min baseline recording, 5 min at 5 m min−1, 9 m min−1, 12 m min−1 and 15 m min−1, and then 2 min at 17–47 m min−1 increasing by 2 m min−1. Mice were run until they are exhausted. Exhaustion was qualified by a mouse sitting on the shocker (1.60 mA, 120 V, 3 Hz) for five consecutive seconds, at which point the shocker was shut off and treadmill schedule stopped. Then, 15–20 min of recovery data were recorded.

Primary myoblast culture and differentiation

Primary myoblasts were isolated from hind limb muscles of 2 month-old WT and TKO male mice. The isolated myoblasts were cultured in myoblast growth media (F-10 media, 20% FBS, 10 ng ml−1 basic fibroblast growth factor, Penicillin-Streptomycin). For differentiation into myotubes, when primary myoblasts were around 95% confluency, differentiation media (DMEM, 2% horse serum) was treated for 5 days. Phase contrast microscope images were taken under an inverted microscope attached to a digital camera during the course of differentiation. For fluorescent imaging, fully differentiated myotubes were incubated with 100 nM Mitotracker CMX-ROS (M7512, Invitrogen) for 30 min at 37 °C. After fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100, the cells were treated with 6.6 µM Alexa Fluor 488-conjugated phalloidin (A12379, Invitrogen) for 45 min and 1 μg mL−1 DAPI for 10 s at room temperature then imaged under Leica SP5 confocal microscope.

Measurements of mitochondrial oxygen consumption

Mitochondrial oxygen consumption rate was measured through the XFe96 Extracellular Flux Analyzer (Seahorse Biosciences) according to the manufacturer’s recommendation. Oxygen consumption rate (OCR, moles min−1) was measured as an index of mitochondrial function. Initially, baseline rates were measured, and then, 1 μM oligomycin, 0.25 μM FCCP and 0.5 μM rotenone/antimycin A (XF cell mito stress test kit from Agilent Technologies, 103015-100) were injected sequentially through the ports of the Seahorse flux assay kit cartridge. The rates were measured at three consecutive time points. A line diagram of the OCR measurements was shown after normalization with baseline rates. Mitochondria-specific respiration rates were also calculated. Basal mitochondrial respiration was derived by subtracting the rotenone rate from the baseline rate, ATP-linked respiration by subtracting the oligomycin rate from the baseline rate, proton leak respiration by subtracting the rotenone rate from the oligomycin rate, maximal mitochondrial respiration by subtracting the rotenone rate from the FCCP rate, and reserve mitochondrial respiration by subtracting the baseline rate from the FCCP rate. All these mitochondrial respiration values were normalized by non-mitochondrial respiration, which is same as the rotenone rate.

C2C12 myoblast culture and differentiation

C2C12 cells were originated from ATCC (CRL-1772) and cultured in DMEM supplemented with 10% FBS and Penicillin-Streptomycin. Cells were tested negative for Mycoplasma contamination in PCR-based analysis using these primers: F: GTGGGGAGCAAA(C/T)AGGATTAGA, and R: GGCATGATGATTTGACGTC(A/G)T. For differentiation, when cells were at 80–90% confluence, growth media was replaced with differentiation media (DMEM, 2% horse serum) for 5 days. Differentiated C2C12 cells were verified through their myotube morphology, and subjected to adenoviral infection.

Adenoviral procedures

Flag-tagged full-length human Sesn2 was cloned into pACCMV-I shuttle vector, and then assembled into the adenoviral backbone at the University of Michigan Vector Core (Ad-SESN2). GFP-expressing adenoviruses (Ad-GFP), constructed in the same way, were used as a negative control. For dose-dependent infection experiments for C2C12 myotubes, total amount of viral particles used for infection was kept constant, and the ratio between Ad-SESN2 and Ad-GFP was proportionally changed according to the infection scheme. At 36 h after infection, harvested cells were subsequently utilized for immunoblotting or quantitative RT-PCR.

Antibodies

dSesn, Sesn1, and Sesn2 antibodies were generated from rabbits and guinea pigs using GST-fusion proteins14,25. All in-house antibodies were affinity purified using PVDF-immobilized target proteins and confirmed through knockout tissue lysates. Commercial Sesn3 antibodies (Abcam, ab97792) were also used to detect Sestrins. Tubulin (T5168) antibodies are from Sigma. Actin (JLA20), Wingless (4D4), type I MHC (BA-D5), type IIA MHC (SC-71) and type IIB MHC (BF-F3) antibodies are from Developmental Studies Hybridoma Bank (DSHB). Phospho-Thr398 Drosophila S6k (9209), phospho-Thr389 S6k (9234), phosphor-Thr172 AMPKα (phospho-Thr184 Drosophila AMPK; 2535), AMPKα (2532), phospho-Ser79 ACC (3661), ACC (3676), Flag (2368), phospho-Ser505 Drosophila Akt1 (4054), phospho-Ser473 mouse Akt1 (9271) and mouse/Drosophila Akt antibodies (4691) are from Cell Signaling Technology. PGC1α (sc-517380), PGC1β (sc-373771), MYH (skeletal muscle myosin heavy chain; sc-32732), MyoD (sc-377460), NDUFS3 (sc-374282), SDHA (sc-390381), SDHB (sc-271548), COX2 (sc-514489), UQCRFS1 (sc-271609), UQCRC2 (sc-390378), ATP5A (sc-136178), ATP5B (sc-55597) and S6K (sc-8418) antibodies are from Santa Cruz Biotechnology. For immunoblotting, antibodies were diluted at 1:200 for Santa Cruz antibodies and at 1:1,000 for all other antibodies. For immunostaining, antibodies were diluted at 1:500 (type IIA MHC antibody) or 1:100 (all other antibodies).

Muscle histology

Whole mouse soleus and gastrocnemius muscles were snap frozen in O.C.T compound (Tissue-Tek) using isopentane-cooled in liquid nitrogen, and cut into 10-µm-thick cryosections. To determine muscle fiber type, muscle cryosections were permeabilized in 0.5% Triton X-100 in PBS, treated in MOM blocking solution (Vector laboratories, BMK-2202), according to the manufacturer’s instructions. The sections were incubated overnight at 4 °C with primary monoclonal antibodies against type I myosin heavy chain (MHC) (BA-D5, mouse IgG2b), type IIA MHC (SC-71, mouse IgG1) and type IIB MHC (BF-F3, mouse IgM). The primary monoclonal antibodies were detected with goat anti-mouse secondary antibodies against IgG2b (Alexa 350, A-21140), IgG1 (Alexa 555, A-21127), and IgM (Alexa 647, A-21238), obtained from Invitrogen. To visualize extracellular matrix, wheat germ agglutin (WGA) lectin conjugated to AlexaFluor 488 (Life Technologies, W11261) was used. Type IIX muscle fibers were detected by the absence of immunofluorescent signal. Fluorescence images were obtained through Nikon A1 confocal microscope. The sections of gastrocnemius were stained for NADH-TR (complex I) and succinate dehydrogenase (SDH, complex II) through the following method. For histochemical detection of mitochondrial complex I activity, sections were incubated for 30 min at 37 °C in freshly prepared 1 mg ml−1 nitroblue tetrazolium and 1 mg ml−1 NADH in 100 mM Tris (pH 7.6) solution. For detecting mitochondrial complex II activity, the sections were dried at the room temperature for 10 min and were rehydrated with PBS (pH 7.2), and then incubated in a complex II assay solution containing 50 mM phosphate buffer (pH 7.4), 50 mM succinic acid, and 0.5 mg mL−1 nitroblue tetrazolium (NBT) at 37 °C in a humidity chamber for 30 min. The sections were washed in distilled water, dried, and then mounted in glycergel mounting medium (Dako). All histological sections were imaged under a light microscope (Meiji).

Transmission electron microscopy

For transmission electron microscopy, samples were fixed in 2.5% glutaraldehyde in 0.1 M Sorensen’s buffer, pH 7.4, overnight at 4 °C. The next day, samples were rinsed twice in Sorensen’s, fixed in 1% osmium tetroxide in Sorensen’s for 1 h, and rinsed in double distilled water. The samples were then dehydrated in ascending concentrations of ethanol, 10 min each, rinsed twice in acetone, and embedded in epoxy resin. Resin blocks were cut to 70 nm ultra-thin sections and stained with uranyl acetate and lead citrate. Sections were imaged on a JEOL 1400 + electron microscope at 80 keV with Hamamatsu ORCA-HR digital camera system.

Contractile force

Contractile properties of skeletal muscle were examined through the following method70. Intraperitoneal injection of tribromoethanol (400 mg kg−1) was used to anesthetize the mice, and anesthesia was maintained by supplemental injections of tribromoethanol throughout the procedure. Contractile properties of gastrocnemius (GTN) muscle were measured in situ. The whole GTN muscle was isolated from surrounding muscle and connective tissue of anesthetized mice, and the distal tendon was severed and secured to the lever arm of a servomotor (model 305B, Aurora Scientific). Muscles were activated via stimulation of the tibial nerve by platinum wire electrodes. Stimulation voltage was adjusted to produce maximum force, typically between 5 and 10 V. With muscles held at optimum length for force production, trains of 0.2 ms stimulus pulses were applied. Pulse frequency was increased until a maximum force was reached. Contractile properties of soleus (SOL) muscles were measured in vitro. Each SOL muscle was removed from the animal and placed in a horizontal bath containing buffered mammalian Ringer solution (137 mM NaCl, 24 mM NaHCO 3 , 11 mM glucose, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , and 0.025 mM turbocurarine chloride) maintained at 25 °C. The solution was bubbled with 95% O 2 /5% CO 2 to maintain pH 7.4. One tendon was tied to a force transducer (model BG-50, Kulite Semiconductor Products Inc.) and the other tendon was tied to a fixed post. Muscles were stimulated between two platinum plate electrodes, and a maximum force was measured as described above for GTN muscles. After all force measurements, muscle mass was measured and total fiber cross-sectional area (CSA) was calculated by dividing the muscle mass by the product of fiber length (determined from previously established ratios of optimum muscle length to fiber length) and muscle density, 1.06 g per cm2. Maximum specific force (SOL, kN per m2; GTN, N per cm2) was calculated for each muscle by dividing maximum force by CSA.

Immunoblotting

For immunoblotting, tissue lysates were boiled in 1X SDS sample buffer for 5 min, separated by SDS-PAGE, transferred to PVDF membranes and probed with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence was detected using LAS4000 (GE) systems. Uncropped immunoblot images were provided as a Source Data file.

Immunostaining of imaginal discs

Third instar wandering-stage larvae of the indicated genotypes were collected, rinsed and dissected in phosphate-buffered saline (PBS) for immunostaining14. Imaginal disc complexes were fixed in Brower Fix (0.15 M PIPES pH 6.9, 3 mM MgSO 4 , 1.5 mM EGTA, 1.5% NP-40) mixed with one-third volume of 8% methanol-free formaldehyde for 3 h at 4 °C. After washing in PBT (PBS with 0.1% Tween-20), the tissues were incubated in 1X Western blocking reagent (Roche) diluted in PBT for 1 h at room temperature. The tissues were then incubated overnight at 4 °C with primary antibodies in 1X Western blocking reagent. The tissues were then washed with PBT, and incubated for 3 h at room temperature with fluorophore-conjugated secondary antibodies (Invitrogen) in 1X Western blocking reagent. After washing with PBT, the tissues were rinsed with PBS and mounted in ProLong Gold anti-fade reagent (Invitrogen). Samples were examined under a Nikon A1 confocal microscope.

Quantitative RT-PCR

Total RNA was extracted using the Trizol system (Invitrogen). RNA was treated with DNase I (Thermo Fisher, 18068015) and reverse transcribed using MMLV reverse transcriptase (Thermo Fisher, 28025013) with random hexamers (Thermo Fisher, N8080127). Relative transcript amounts were measured by the StepOnePlus Real Time PCR system (Applied Biosystems), using iQ SYBR Green Supermix (Bio-rad, 1708884). All mRNA expression data were normalized to the Actb level (mouse) or the rp49 level (Drosophila). Following primer pairs were used.

Myod1 F, CCACTCCGGGACATAGACTTG

Myod1 R, AAAAGCGCAGGTCTGGTGAG

Myog F, GAGACATCCCCCTATTTCTACCA

Myog R, GCTCAGTCCGCTCATAGCC

Myf5 F, AAGGCTCCTGTATCCCCTCAC

Myf5 R, TGACCTTCTTCAGGCGTCTAC

Myh7 (Mhc-I) F, ACTGTCAACACTAAGAGGGTCA

Myh7 (Mhc-I) R, TTGGATGATTTGATCTTCCAGGG

Myh2 (Mhc-IIa) F, AAGTGACTGTGAAAACAGAAGCA

Myh2 (Mhc-IIa) R, GCAGCCATTTGTAAGGGTTGAC

Myh1 (Mhc-IIx) F, GCGAATCGAGGCTCAGAACAA

Myh1 (Mhc-IIx) R, GTAGTTCCGCCTTCGGTCTTG

Myh4 (Mhc-IIb) F, TTGAAAAGACGAAGCAGCGAC

Myh4 (Mhc-IIb) R, AGAGAGCGGGACTCCTTCTG

Pgc1a F, TATGGAGTGACATAGAGTGTGCT

Pgc1a R, CCACTTCAATCCACCCAGAAAG

Pgc1b F, TCCTGTAAAAGCCCGGAGTAT

Pgc1b R, GCTCTGGTAGGGGCAGTGA

Ppara F, AGAGCCCCATCTGTCCTCTC

Ppara R, ACTGGTAGTCTGCAAAACCAAA

Esrra F, CTCAGCTCTCTACCCAAACGC

Esrra R, CCGCTTGGTGATCTCACACTC

Tfam F, ATTCCGAAGTGTTTTTCCAGCA

Tfam R, TCTGAAAGTTTTGCATCTGGGT

Cytc F, CAGCTTCCATTGCGGACAC

Cytc R, GGCACTCACGGCAGAATGAA

Ckmt F, ACACCCAGTGGCTATACCCTG

Ckmt R, CCGTAGGATGCTTCATCACCC

Cox5a F, GCCGCTGTCTGTTCCATTC

Cox5a R, GCATCAATGTCTGGCTTGTTGAA

Mtco2 F, AATTGCTCTCCCCTCTCTACG

Mtco2 R, GGTTTTAGGTCGTTTGTTGGGAT

Cpt1b F, GCACACCAGGCAGTAGCTTT

Cpt1b R, CAGGAGTTGATTCCAGACAGGTA

Oxct1 F, CATAAGGGGTGTGTCTGCTACT

Oxct1 R, GCAAGGTTGCACCATTAGGAAT

Mdh1 F, TTCTGGACGGTGTCCTGATG

Mdh1 R, TTTCACATTGGCTTTCAGTAGGT

Idh3a F, TGGGTGTCCAAGGTCTCTC

Idh3a R, CTCCCACTGAATAGGTGCTTTG

Actb F, CAAAAGCCACCCCCACTCCTAAGA

Actb R, GCCCTGGCTGCCTCAACACCTC

dPGC1 F, GGATTCACGAATGCTAAATGTGTTCC

dPGC1 R, GATGGGTAGGATGCCGCTCAG

rp49 F, ACGTTGTGCACCAGGAACTT

rp49 R, CCAGTCGGATCGATATGCTAA

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.