Metformin and phenformin (Sigma) were dissolved in water with the stock solutions as 1 M and 0.2 M respectively. AICAR (LC Laboratories) was dissolved in water at 50 mM. Rotenone (Sigma) and rapamycin (LC laboratories) were dissolved in ethanol to make 2.5 mM and 10 mM stock solutions, respectively. For transfection, Lipofectamine 3000 was used according to the instructions of manufacturer (Invitrogen). For western blotting, we used the anti-TPR antibody (NB100-2866, Novus Biologicals), the self-generated anti-RagC antibody (), and antibodies against p-S244/240 S6 (#2215), total S6 (#2217), mTOR, TSC2, Raptor as well as RagA from Cell Signaling Technology, and FLAG-Tag from Sigma. For immunostaining of RagC, the same self-made Rabbit RagC mAb and the corresponding secondary Anti-Rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate, Cell Signaling Technology) was used, while Alexa Fluor 594 anti-nuclear pore complex proteins antibody, mAb414, was used to stain NPCs (BioLegend).

C8161 cells (ATCC) were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Life Technologies) in a 5% CO2 atmosphere at 37°C. All other cell lines used in this study were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% FBS and 1% PS in a 5% CO2 atmosphere at 37°C. For western blotting and cell fractions, HEK293E cells (a gift from Dr. Ramnik Xavier) were used. For cell viability measurements, C8161 (ATCC), MeWo (ATCC) and PANC1 (a gift from Dr. Nabeel Bardeesy) cells were used. For nuclear permeability testing, C8161 and HeLa cells were used. For GST-RagC W /FLAG-RagA X expression, HEK293T cells (ATCC) were used. For ACAD10 mRNA level determination, HEK293E and C8161 cells were used. For immunofluorescence assays, HeLa cells (ATCC) were used.

All nematode strains were grown at 20°C, and maintained following standard procedures (). N2 Bristol was used as the wild-type strain. The listed mutant strains were used: MGH325 CeACAD10(gk463343) II 4x, MGH275 aak-1(tm1944) III 4X, MGH274 aak-2(ok524) X 4X, MGH276 aak-1(tm1944) III;aak-2(ok524) X 4X, MGH326 npp-3(alx43) II 3X, and MGH327 npp-21(alx44) II 4X. The following transgenic lines were used: MGH249 alxIs19[CeACAD10p::CeACAD10::mRFP3-HA myo-2p::GFP] 8X, MGH328 alxEx90[CeACAD10p::CeACAD10::mRFP3-HA myo-2p::GFP], MGH322 npp-3(alx43);alxIs19 3X, MGH323 npp-21(alx44);alxIs194X, MGH277 aak-2(ok524);alxIs19 4X, CF1553 muIs84 [(pAD76) sod-3p::GFP + rol-6] 3X and MGH50 mgIs48[ges-1p::MITO::GFP] 0X, LG326 skn-1(zu169) IV; geIs7[skn-1b::GFP].

Method Details

Feeding RNAi in C. elegans Pino et al., 2013 Pino E.C.

Webster C.M.

Carr C.E.

Soukas A.A. Biochemical and high throughput microscopic assessment of fat mass in Caenorhabditis elegans. The RNAi clones used in this study were all isolated from a genome-wide E. coli feeding RNAi library and fed to C. elegans as described previously (). Synchronous C. elegans were obtained by bleach treatment of gravid adults, and after 72 hr of RNAi feeding, animals were harvested and examined as day-1 adults unless noted otherwise. For the RNAi screen of metformin response genes, the RNAi bacteria were seeded onto 96-well plates using NGM agarose supplemented with 5 mM IPTG (US Biologicals) and 100 μg/mL carbenicillin (Sigma), free liquid was driven off in a laminar flow hood, and RNAi plates were incubated at room temperature overnight. The following day, the indicated dose of metformin was added into 96-well RNAi plates and free liquid was driven off in a laminar flow hood. Synchronous populations of L1 stage, wild-type worms were dropped onto 96-well RNAi plates with or without metformin, and imaged directly from the RNAi plates after 3 days exposure to drugs using a Leica DM6000 microscope at a magnification of 40X. Imaged worms were scored based on their stages and sizes compared to controls.

Quantitative RT-PCR To quantify changes in mRNA abundance in nematode and human cells, total RNA was extracted using TRIzol (Invitrogen) according to manufacturer instructions. RNA was treated with RNase free DNase prior to reverse transcription with the Quantitect reverse transcription kit (QIAGEN). Quantitative PCR was conducted in triplicate using Quantitect SYBR Green PCR reagent (QIAGEN) following manufacturer instructions on a Bio-Rad CFX96 Real-Time PCR system (Bio-Rad). For C. elegans, all assays were performed with 2,000 animals at late L4 stage per sample. Drug treatments were started from synchronous L1 larvae stage and ended as late-L4 stage. Drug treated worms were harvested by washing off of plates and washed an additional 4 times with M9 buffer, allowing worms to settle by gravity between washes. RT-PCR assays for human cells were conducted with 2 X 105 cells post-24 hr drug treatments or 96 hr shRNA transduction. Both worms and cells were flash frozen in liquid nitrogen and kept in −80°C until RNA preparation. The sequences of used primer sets were: for C. elegans: CeACAD10: 5′-GCCATGCTGACAAACAACAT-3′ and 5′-TCAAATCGCATGGAAGTCTG-3′, npp-3: 5′-CCTCGacTTCTCGATCTTCG-3′ and 5′-GTTGAActTTTGCATCAACACAC-3′, npp-21: 5′-ACAGCACGCCAAGAATCAG-3′ and 5′-TCCGTATtcTCGTTGCGATA-3′, act-1: 5′-TGCTGATCGTATGCAGAAGG-3′ and 5′-TAGATCCTCCGATCCAGACG-3′, daf-15: 5′-CTGAGAAggCATGTATGCATC-3′ and 5′-ATGGGCAGAATGGATTTGAA-3′; for human cells: ACAD10: 5′-CGAGTGGCAAAGCAGTTCC-3′ and 5′-CCATGCAGGACTCCACAATCA-3′, TPR: 5′-AACGCCAGCGTGAGGAATATG-3′ and 5′-ATTACGTGGTTACCCCTTGCT-3′, NUP205: 5′-GTACTGGGATGGAAAGCGATG-3′ and 5′-GCTCTGGACTGAGTTCTAGGG-3′, NUP153: 5′-CAGGGGCCAATTAAGCCTTAC-3′ and 5′-ACCTCGCTTGTGTCTGTTGAA-3′, NUP98: 5′-CTCCACCACTAATTCAGGCTTT-3′ and 5′-GAGGCTGGTAGTCTGCTGATT-3′, NUP214: 5′-TGACTCCCCTGAGGAATTGC-3′ and 5′-GCGAAGACCAGACCATATTTGTT-3′, beta-actin: 5′-CATGTACGTTGCTATCCAGGC-3′ and 5′-CTCCTTAATGTCACGCACGAT-3′. Expression levels of tested genes were presented as normalized fold changes to the mRNA abundance of act-1 for worms and beta-actin for human cells respectively by the ΔΔCt method.

Lentiviral shRNA Infection Yuan et al., 2012 Yuan M.

Pino E.

Wu L.

Kacergis M.

Soukas A.A. Identification of Akt-independent regulation of hepatic lipogenesis by mammalian target of rapamycin (mTOR) complex 2. Lentiviral shRNA constructs based in pLKO1 were obtained from the Broad Institute of Harvard and MIT Genetic Perturbation Platform and are commercially available from Sigma. shRNA delivery was conducted as previously published (). shRNA knockdown cells post-24 hr-selection with 2 μg/ml puromycin were treated in medium containing 0.5 μg/ml puromycin with or without drugs for the indicated times followed by western blotting or cell viability measurement. The knockdown efficiency of targeted genes by shRNA in different cell lines was examined by RT-PCR for mRNA levels under identical experimental conditions at the identical time of experiments.

Western Blotting Cell lysates were prepared immediately following the indicated treatments by boiling in 0.5% SDS for 10 min, followed by incubation on ice. Protein concentration was determined using the Pierce BCA assay (Thermo Fisher). SDS-PAGE was conducted on the same day that lysates were made, followed by electrophoretic transfer to nitrocellulose membrane at 50 V for 16 hr at 4°C. Immunoblots were performed according to primary antibody manufacturers’ protocols. For immunodetection of primary antibodies, goat-anti-rabbit-HRP conjugate (GE Healthcare) was used at 1:5,000 in 5% BSA dissolved in TBST, and HRP was detected using West-Pico chemiluminescence substrate (Thermo Pierce). The western blot results shown are representatives of at least two independent biological replicates.

Immunofluorescence HeLa cells were plated on glass coverslips in 6 well cell culture plates grown to about 50% confluence. Cells were treated with vehicle and 1 mM phenformin for 24 hr, then washed twice in PBS and fixed with 4% paraformaldehyde in PBS for 15 min. Cells were rinsed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 min. After rinsing with PBS, cells were blocked in 3% BSA/PBS for 30 min and then incubated in 3% BSA with anti-RagC antibody overnight. Cells were washed three times in PBS and then incubated in 3% BSA with Anti-Rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) for 1 hr. For NPC immunostaining with mAb414 antibody, the experiment was done exactly following the instructions from the manufacturer (BioLegend). Cells were then rinsed three times for 5 min each in PBS and mounted using ProLong Diamond Antifade mounting medium with DAPI (Life Technologies). Images were captured using a Leica DM6000 microscope with a 63X oil immersion objective.

Body Size Determination of C. elegans Animals, post-RNAi feeding, drug treatment, or combinations of both for 72 hr, were paralyzed in M9 buffer with 1 mg/ml levamisole (Sigma), and then imaged at 5X magnification on a Leica DM6000 microscope. Maximal, longitudinal cross-sectional area of the imaged C. elegans was determined by using MetaMorph software for a minimum of 27 animals per condition in each experiment. Results of single experiments are shown. Each experiment was performed at least twice, and results were consistent between experiments.

Creation of the CeACAD10 Fusion and Promoter Transgenic Lines 2 UV-C using a Stratalinker 2400 UV crosslinker (Stratagene, La Jolla, CA, USA) and screened for the successfully integrated lines, while the promoter only transgenic was used directly for expression tests post-metformin treatment. For the CeACAD10 reporter ( Figure 1 G), the entire genomic sequence of CeACAD10 locus (1367 bp), including introns and exons, plus 2418 bp of promoter were amplified and cloned into a modified version of the commercial Fire vector pPD95.77 (the GFP region was swapped with monomeric red fluorescent protein (mRFP) with a C-terminal HA epitope tag) at the SbfI and SalI sites. The following cloning primers were used for the fusion reporter: forward: 5′-AGCCTGCAGGACCGTAGCTCCAGTGTCGATT-3′ and reverse: 5′-AGCCTGCAGGTCCACAAAAATTGCTCATTGC-3′. The same forward primer above was used for cloning the promoter only transgenic line ( Figure 2 G), while the reverse one was: 5′-CCGGGTACCTGCTGGGTCATAGTCATTTTG-3′. Both fragments were injected at 20 ng/μl into the gonad of wild-type adult animals. The CeACAD10 reporter was integrated into the genome with 250 J/mUV-C using a Stratalinker 2400 UV crosslinker (Stratagene, La Jolla, CA, USA) and screened for the successfully integrated lines, while the promoter only transgenic was used directly for expression tests post-metformin treatment.

Isolation of Metformin Suppressor Mutants Kim et al., 2008 Kim E.

Goraksha-Hicks P.

Li L.

Neufeld T.P.

Guan K.L. Regulation of TORC1 by Rag GTPases in nutrient response. The reporter line was mutagenized with ethyl methanesulfonate (EMS, Sigma), and the resultant F2 generation was screened on plates supplemented with 50 mM metformin for suppressors of CeACAD10 expression. Whole genome Illumina sequencing was employed followed by analysis and variant calling in Galaxy () to identify causal mutations. Causal mutations were confirmed by Sanger sequencing and RNAi.

CeACAD10 Reporter Fluorescence Intensity Analysis Pino et al., 2013 Pino E.C.

Webster C.M.

Carr C.E.

Soukas A.A. Biochemical and high throughput microscopic assessment of fat mass in Caenorhabditis elegans. Animals of the reporter line in either wild-type or mutant background were treated with drugs or RNAi for indicated times, paralyzed with 1 mg/ml of levamisole, and then imaged in 96-well format with a Leica DM6000 microscope outfitted with a mCherry filter set and MMAF software. Image analysis was conducted with MATLAB (MathWorks, Inc.) as described previously (). All RFP or GFP intensity tests were carried out in biological triplicate or quadruplicate with at least 30 animals tested per replicate.

ATP Measurement 500 synchronized young adult worms were collected in M9 buffer and washed three times. Worm pellets were treated with three freeze/thaw cycles and boiled for 15 min to release ATP and destroy ATPase activity and then spun at 4°C at 12,000 × g for 10 min. ATP content was measured with an ATP detection kit according to the manufacturer’s instructions (ThermoFisher, A22066), and normalized to total protein content determined by BCA assay.

SKN-1 Chromatin Immunoprecipitation Synchronous L4 animals ( ∼6,000 per plate x 8 large plates) were generated from three independent egg preps of SKN-1::GFP transgenic animals. Animals were washed in M9 with 0.005% Triton X-100 three times, washed once with M9 with 0.005% triton with 1% formaldehyde, then frozen drop wise in 3 pellet volumes of the same cross-linking buffer (M9 plus 0.005% Triton X-100 plus 1% formaldehyde) in liquid nitrogen and kept at −80C. Pea-size frozen pellets were crushed in a dry-ice-chilled mortar and pestle, and ground into powder until worm carcasses are disturbed. After thawing, animals were formaldehyde crosslinked at room temperature for 20 min, followed by quenching with 125 mM glycine (final concentration) at room temperature for 5 min. Pellets were washed three times in ice cold PBS, spinning each time 2 min 4400 rpm between washes at 4C. Pellets were then resuspended in 2 pellet volumes ice cold FA lysis buffer (50 mM HEPES: adjust pH to 7.5 with KOH, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.2% Sodium Lauryl Sarcosine (AKA Sarkosyl; sodium lauryl sarcosinate), and 2 mM PMSF added immediately before use. Sonication was conducted in a Qsonica Q800R with total sample volume not exceeding 180ul per tube with total sonication time of 25 min, amplitude 70%, 30 s pulses on alternating with 30 s off. The resulting chromatin was cleared by centrifugation at 14,000rpm (21,000 g) for 15 min at 4C. The supernatant was retained and protein quantified. One mg of chromatin was used for chromatin immunoprecipitation diluted to 1 mL final volume with no detergent FA Lysis buffer (50 mM HEPES: adjust pH to 7.5 with KOH, 150 mM NaCl, 1 mM EDTA, and 2 mM PMSF added immediately before use). Triton X-100 was added from 25% stock to 1% final concentration. Each 1 mL chromatin was pre-cleared with 30 μl of magnetic protein G Sepharose at 4°C for 1 hr. Fifty microliters were removed and used to assess input DNA. Pre-blocked 30uL magnetic protein G-Sepharose (with 1 mg/ml BSA and 0.3 mg/ml of salmon sperm DNA in FA lysis buffer + 1% Triton X-100) were added per IP (primary antibody and negative control IgG antibody) along with 5 μg of A-11120 mouse anti-GFP monoclonal antibody (Invitrogen) or IgG2a isotype control (02-6200, Invitrogen). Chromatin IPs were incubated overnight at 4°C on a Nutator. Beads were washed five times with 1 mL of no detergent FA lysis buffer + 1% Triton X-100, followed by 1 mL of no detergent FA lysis buffer +1% Triton X-100 + 0.5 M NaCl (3 min wash end over end rotator). Beads were washed with 1 mL TE and immune complexes were eluted with 100 μL ChIP elution buffer 50 mM Tris-Cl, pH 7.5, 10 mM EDTA, 1% SDS 10 min at 65°C. An equal volume of RNase A mix (50 mM Tris pH 8.0 99.5 parts to 0.5 part RNase A (Sigma R4642) in 50% glycerol (stock 33 mg/mL)) was added to eluted ChIP and input samples and incubated for 1 hr at 37°C. Thereafterer 2 μL per 100 μL of 20mg/mL ChIP Proteinase K (4 μL for 200uL of total volume, chromatin plus ChIP elution buffer; PK stock in water +50% glycerol, Sigma P4850) was added to each sample followed by incubation 2 hr at 55C and then 6 hr to overnight at 65C to digest protein and reverse cross links. DNA was cleaned in a QIAGEN PCR clean up column and eluted in 50 μL elution buffer. One half μL was added to each qPCR reaction. Signal minus IgG background was expressed relative to the amount of input DNA after calculating the amount of each qPCR reaction from an 8-point standard curve. Primers used for qPCR of ACAD10 genomic loci were as follows: ACAD10p −460nt F, tttttgaacattttggttttgacc; ACAD10p −460nt R, ttccctcgctggaactgaac; ACAD10p −105nt F, gaccaactcgttcagttttgatg; ACAD10p −105nt R, tttgctgggtcatagtcattttg; ACAD10 3′UTR +1.7kb F, tattatgcggcggaatgtcac; ACAD10 3′UTR +1.7kb R, tgcaagcagtgtgattacttgg.

Longevity Assay Cabreiro et al., 2013 Cabreiro F.

Au C.

Leung K.Y.

Vergara-Irigaray N.

Cochemé H.M.

Noori T.

Weinkove D.

Schuster E.

Greene N.D.

Gems D. Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism. Han et al., 2016 Han S.K.

Lee D.

Lee H.

Kim D.

Son H.G.

Yang J.S.

Lee S.V.

Kim S. OASIS 2: online application for survival analysis 2 with features for the analysis of maximal lifespan and healthspan in aging research. Lifespan analysis was conducted at 20°C according to a protocol modified from our previous and other’s procedures (). Briefly, synchronized L1 animals were seeded onto the standard nematode growth media (NGM) plates until L4 stages. On day 0, 30–50 L4 worms per plate (3 plates, 90–150 worms in total per condition) were transferred onto NGM (for mutants) or RNAi plates (for RNAi) with or without metformin co-treatment. All plates were supplemented with 100 μM 5-fluorodeoxyuridine (FUdR) solution to suppress progeny production. Statistical analysis was performed with online OASIS2 resources ().

Cell Fractionation Suzuki et al., 2010 Suzuki K.

Bose P.

Leong-Quong R.Y.

Fujita D.J.

Riabowol K. REAP: A two minute cell fractionation method. Cell fractionation was carried out following a rapid and efficient protocol () to avoid possible loss of any nucleocytoplasmic shuttling proteins from the nucleus during the time of isolation, with the following modifications. In this study sub confluent HEK293E cells untreated or treated with 24 hr on 6-well plates were washed 3 times with ice cold PBS prior to fractionation with 0.1% NP40 in PBS. Total lysates, cytosol and nuclear fractions were collected at each step of fractionation for western blot analysis.

Cell Viability Measurement Yuan et al., 2013 Yuan P.

Ito K.

Perez-Lorenzo R.

Del Guzzo C.

Lee J.H.

Shen C.H.

Bosenberg M.W.

McMahon M.

Cantley L.C.

Zheng B. Phenformin enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma. Cell viability analysis was conducted as previously described (). In brief, shRNA knockdown cells were seeded onto 96-well plates at a density of 800–1,000 cells per well, and drug treatment was initiated the following day. After 3 days of drug treatment, the viability measurement was carried out with the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions.

Nuclear Permeability Assay C8161 cells were plated with 3 X 105 cells per well onto sterilized coverslips in the base of 6-well plates 24 hr before drug treatment. After 24 hr of drug treatment, cell membranes were permeabilized with 40 μg/ml digitonin in freshly made transport buffer (TB) containing 20 mM HEPES, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 0.5 mM EGTA, 2 mM DTT and protease inhibitor cocktail (Sigma) at room temperature. Digitonin permeabilization times were optimized by using trypan blue (Life Technologies) staining in C8161 cells (10 min) to efficiently permeabilize plasma membranes while keeping the nuclear envelope intact as indicated by full exclusion of fluorescein isothiocyanate (FITC)-Dextran 500000-Conjugate (Sigma). Immediately after semi-permeabilization, cells were rinsed twice with TB and incubated with fluorescent labeled dextran mixtures at the concentration of 2 mg/ml of each dextran for 20 min, including FITC-Dextran 500000-Conjugate mixed with Tetramethylrhodamine isothiocyanate (TRITC)-Dextran average molecular weight 65,000–85,000 (Sigma) for C8161 cells. Then, coverslips were inverted onto a 15 μl drop of Antifade mounting medium made with 1X PBS, 90% glycerol, and 2% n-propyl gallate (Sigma) for imaging. Images were taken from randomly distributed fields of the coverslips using a Leica DM6000 microscope outfitted with a standard GFP filter set or mCherry filter set and MMAF software at a 63X oil immersion objective. Scale bars were added using ImageJ software. The fluorescence intensities of intranuclear area and extranuclear background for each nucleus were measured using ImageJ, and the intensity ratio of intranuclear and extranuclear levels was calculated to index nuclear permeability.

RagC Proteomic Analysis Flag-tagged RagC and RagA were co-transfected into HEK293T cells in order to prepare four samples: 1) control (no drug) total RagC, 2) total RagC subjected to treatment with 1 mM phenformin, 3) control (no drug) cytoplasmic RagC, and 4) control (no drug) nuclear RagC. Flag-tagged RagC was immunopurified by large-scale immunoprecipitation with anti-Flag M2 magnetic beads (Sigma), eluted with Flag peptide and separated by SDS-PAGE. The 4 gel bands were in-gel digested with trypsin and analyzed by a Q-Exactive mass spectrometer, which provided high-resolution MS peptide mass measurement and high-resolution MS/MS peptide sequencing. We searched the 4 data files with both our in-house Sequest-based platform, as well as the commercial software PEAKS in attempt to identify modifications in RagC. Overall sequence coverage was above 60% for phenformin and control, 90% for cytoplasmic and 50% for nuclear fractions, which correlated well with gel band intensity. For our in-house platform, we used a wide mass tolerance no enzyme search and a single protein database. Identified peptides were limited to fully tryptic peptides having a ppm tolerance of less than 10 ppm and a cross-correlation score of greater than 2.5. We specifically searched for oxidation, mono-, di- and tri-methylation, acetylation and phosphorylation with our in-house software and 485 different modifications using the PEAKS software.