Mice Martin et al., 1998 Martin F.

van Deursen J.M.

Shivdasani R.A.

Jackson C.W.

Troutman A.G.

Ney P.A. Erythroid maturation and globin gene expression in mice with combined deficiency of NF-E2 and nrf-2. fl/fl (WT) mice with Tsc1fl/fl | Albumin-Cre+/− (LTsc1KO) mice as previously described ( Harputlugil et al., 2014 Harputlugil E.

Hine C.

Vargas D.

Robertson L.

Manning B.D.

Mitchell J.R. The TSC complex is required for the benefits of dietary protein restriction on stress resistance in vivo. Yang et al., 2008 Yang G.

Wu L.

Jiang B.

Yang W.

Qi J.

Cao K.

Meng Q.

Mustafa A.K.

Mu W.

Zhang S.

et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. All rodent experiments were performed with the approval of the appropriate institutional animal care and use committee. Male B6D2F1 hybrid mice 8-10 weeks of age purchased from The Jackson Laboratory were used for all experiments unless otherwise indicated. Male and female NRF2 KO and littermate control mice on a mixed 129/C57BL6 background generated previously () were bred in our facility and used at the age of 8-10 weeks. LTsc1KO and littermate control animals were generated by crossing Tsc1(WT) mice with Tsc1| Albumin-Cre+/− (LTsc1KO) mice as previously described (). 8 week old male CGL WT and KO mice were generated as previously described ().

Preconditioning Regimens Dietary Miller et al., 2005 Miller R.A.

Buehner G.

Chang Y.

Harper J.M.

Sigler R.

Smith-Wheelock M. Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance. Mice were given ad libitum (AL) access to food and water unless otherwise indicated. Experimental diets were based on Research Diets D12450B with approximately 18% of calories from protein (hydrolyzed casein or individual crystalline amino acids (Ajinomoto) in the proportions present in casein), 10% from fat and 72% from carbohydrate. AL food intake per gram of body weight was monitored daily for several days and used to calculate 50% dietary restriction (DR) based on initial animal weights. Protein free diets were kept isocaloric by replacing casein/amino acids with an equal weight of sucrose and provided either AL or 35% DR as indicated. MetR diets containing 1.5g Met/kg food and lacking Cys () were provided AL. Met and Cys supplementation back to AL levels in DR diets was achieved by raising Met content from 2.5 to 5g/kg of food, and Cys content from 2 to 4g/kg food. Animals were fed daily with fresh food between 6pm and 7pm. Pharmacological Parnell et al., 2010 Parnell S.E.

Sulik K.K.

Dehart D.B.

Chen S.Y. Reduction of ethanol-induced ocular abnormalities in mice through dietary administration of N-acetylcysteine. Ergul et al., 2010 Ergul Y.

Erkan T.

Uzun H.

Genc H.

Altug T.

Erginoz E. Effect of vitamin C on oxidative liver injury due to isoniazid in rats. Calfee-Mason et al., 2002 Calfee-Mason K.G.

Spear B.T.

Glauert H.P. Vitamin E inhibits hepatic NF-kappaB activation in rats administered the hepatic tumor promoter, phenobarbital. NAC was supplemented at a daily dose of ∼600mg/kg/day, with ∼400mg/kg/day in the food and ∼200mg/kg/day in the drinking water (). Antioxidant vitamins were supplemented in the food at 1000 mg/kg/day VitC () and 250 mg/kg/day VitE (). NAC and VitC&E supplementation was halted 16-24 hr prior to organ harvest and/or induction of IRI. NaHS (1mM final) or GYY4137 (260 μM final) were placed in the drinking water 7 days prior to hepatic IRI with fresh water changes occurring every day for NaHS and on day 3 for GYY4137. PAG (10mg/kg in saline) and NaHS (3-5mg/kg in saline) were administered by IP injection. Adenoviral-Mediated Gene Delivery Overexpression of CGL was accomplished by IV injection of 1010 PFUs of an adenovirus-type 5 (dE1/E3) containing the CMV promoter driving expression of the mouse CGL gene (Ad-mCTH/CGL, GenBank RefSeq BC019483, ADV-256305 Vector Biolabs) or the negative control virus Ad-CMV Null (1300 Vector Biolabs) 7-days prior to hepatic IRI.

IRI and Measurements of Damage Peng et al., 2012 Peng W.

Robertson L.

Gallinetti J.

Mejia P.

Vose S.

Charlip A.

Chu T.

Mitchell J.R. Surgical stress resistance induced by single amino Acid deprivation requires gcn2 in mice. Warm hepatic or renal ischemia reperfusion injury was induced as previously described (). Briefly, mice were anesthetized with isoflurane and body temperature maintained on a circulating heated water pad. Following laparotomy, a microvascular clamp (Roboz) was placed over the portal triad for hepatic ischemia for 35min, or over each renal pedicle for bilateral renal ischemia for 30min, followed by clamp removal and wound closure. Serum was collected from small volumes of tail blood for up to 24hrs after hepatic injury and up to 48hrs after renal injury. Liver damage was assessed by kinetic measurements of serum ALT, AST (Infinity Reagents, Thermo Scientific) and LDH (Pointe Scientific) in a microtiter plate reader (Biotek Synegy2) and confirmed on a different platform (Piccolo Liver Plus discs, Abaxis). Data for ALT, AST, and/or LDH were normalized to the experimental control for each time point, usually the AL WT group, and time points post reperfusion were combined into one value of liver damage per animal. 24 hr post reperfusion, the left liver lobe was harvested and formalin fixed. Images were taken via digital camera and shown at 1x magnification. Fixed lobes were then paraffin embedded, cut into sections and stained with hematoxylin and eosin for confirmation of liver damage. Representative images are shown with 250 μm scale bars.

Metabolic Parameters Hu et al., 2005 Hu T.

Foxworthy P.

Siesky A.

Ficorilli J.V.

Gao H.

Li S.

Christe M.

Ryan T.

Cao G.

Eacho P.

et al. Hepatic peroxisomal fatty acid beta-oxidation is regulated by liver X receptor alpha. Lazarow, 1981 Lazarow P.B. Assay of peroxisomal beta-oxidation of fatty acids. Mouse weights and % fat mass were obtained using an electronic scale and live animal MRI-based body composition analysis (Echo MRI). Blood glucose was measured from fresh tail blood with an Easy Step Glucometer (Home Aide Diagnostics). True serum triglycerides were calculated from measurements of free glycerol and total glycerol after lipolysis using the Serum Triglyceride Determination Kit (Sigma). Liver RONS and GSH were analyzed from left hepatic lobes normalized by wet weight using OxiSelect In Vitro Green Fluorescence ROS/RNS Assay Kit (Cell Biolabs) and the fluorimetric Glutathione Assay Kit (Sigma) according to the manufacture’s recommendations. Peroxisome β-oxidation of lipids was performed on freshly harvested liver (). Briefly, 100mg of liver was placed into 900uL of ice-cold 0.25M sucrose, homogenized on ice, and centrifuged at 600 g for 10min. 450uL of was mixed with 50uL of 10% Triton X-100 (Sigma) on ice, from which 5uL was mixed with 200uL of reaction mix in a 96-well plate and incubated at 37°C for one minute followed by ± addition of 2uL of palmitoyl-CoA. Peroxisomal lipid oxidation was proportional to the rate of NADH formation in the presence of cyanide to block mitochondrial NADH oxidation. Liver metabolites normalized to total protein content were measured via mass spectrometry at the Beth Israel Deaconess Medical Center Core Facility.

Gene Expression Analysis by qPCR and Western Blot Total RNA was isolated from tissues and cells using miRNeasy Mini Kit (QIAGEN) and cDNA synthesized by random hexamer priming with the Verso cDNA kit (Thermo). qRT-PCR was performed with SYBR green dye (Lonza) and TaqPro DNA polymerase (Denville). Fold changes were calculated by the ΔΔC t method using Hprt and/or Rpl13 genes as standards, and normalized to the experimental WT AL control. Primer sequences are as follows: CGL For: TTGGATCGAAACACCCACAAA Rev: AGCCGACTATTGAGGTCATCA; CBS For: GGGACAAGGATCGAGTCTGGA Rev: AGCACTGTGTGATAATGTGGG; HO-1 For: AAGCCGAGAATGCTGAGTTCA Rev: GCCGTGTAGATATGGTACAAGGA; NQO-1 For: AGGATGGGAGGTACTCGAATC Rev: AGGCGTCCTTCCTTATATGCTA; GSTA4 For: TGATTGCCGTGGCTCCATTTA Rev: CAACGAGAAAAGCCTCTCCGT; GSTM4 For: AGCTCACGCTATTCGGCTG Rev: GCTCCAAGTATTCCACCTTCAGT; GSTT3 For: GGATGGGGACTTCGTCTTGG Rev: TCAGGAGGTACGGGCTGTC; LCAD For: TCTTTTCCTCGGAGCATGACA Rev: GACCTCTCTACTCACTTCTCCAG; Cyp4a14 For: TTTAGCCCTACAAGGTACTTGGA Rev: GCAGCCACTGCCTTCGTAA; HPRT For: TTTCCCTGGTTAAGCAGTACAGCCC Rev: TGGCCTGTATCCAACACTTCGAGA; RPL13 For: TTCGGCTGAAGCCTACCAGAAAGT Rev: TCTTCCGATAGTGCATCTTGGCCT; SOD1 For: GGGACAATACACAAGGCTGT Rev: GCCAATGATGGAATGCTCTC; SOD2 For: GCTTGGCTTCAATAAGGAGC Rev: TGAAGGTAGTAAGCGTGCTC; Catalase For: CGGTAGCTGTGAACTGTCCCTACCG Rev: CTCTGGTGCGCTGAAGCTGT. For protein expression analysis, tissues were homogenized with passive lysis buffer (Promega), normalized for protein content, boiled with SDS loading buffer and separated by SDS-PAGE. Proteins were transferred to PVDF membrane (Whatman) and blotted for CGL (ab151769 Abcam), CBS (ab135626 Abcam) and Actin (13E5 Cell Signaling) and secondarily with HPRT conjugated anti-rabbit antibody (Dako).

H 2 S Measurements Lead Sulfide Method on Intact Tissues For detection of H 2 S production capacity from intact fresh tissue, 100mg of fresh liver and/or kidney tissue was placed in a 1.5mL centrifuge tube containing 750 μl of 10mM Cys (Sigma) and 10 μM Pyridoxal 5′-phosphate (PLP) (Sigma) in PBS. A rectangular lead acetate H 2 S detection strip (Sigma) was wedged into the lid of the microcentrifuge tube so as to avoid contact with the supernatant. Closed tubes were incubated 2-5 hr at 37°C until visible, but not oversaturated, lead sulfide darkening of the paper occurred. Lead Sulfide Method on Lysates or Live Cells For detection of H 2 S production capacity from homogenized tissues, flies or worms, samples were homogenized in passive lysis buffer (Promega) followed by several rounds of flash freezing/thawing. Protein content and volume were normalized via BCA Kit (Thermo). 100-300 μg of protein was added to a final reaction in 96-well format containing 10mM Cys and 10 μM PLP for mammalian tissue, 2mM for worm and 6mM for flies. 6x4 inch pieces of lead acetate paper, made by soaking 703 size blotting paper (VWR) in 20mM lead acetate (Sigma) and then vacuum drying, were placed over the 96-well dish and incubated for 2-24 hr at 37°C until lead sulfide was detected but not saturated. For detection of H 2 S production in live Hepa1-6 cells grown in 96-well plates, growth media was supplemented with 10mM Cys and 10 μM PLP, and a lead acetate paper placed over the plate for 2-24 hr of further incubation in a CO 2 incubator at 37°C. Lead Sulfide Method in Live Yeast Culture Lead acetate paper squares were placed on the bottom of the flask stopper at the time of inoculation of yeast in media containing 2% or 0.5% glucose. H 2 S Detection with Microsulfide Probe A micro-sulfide ion electrode probe (Lazar Research Laboratories) and volt meter (Jenco) were used to measure H 2 S production capacity in extracts in vitro and endogenous hepatic H 2 S levels in vivo. The probe was calibrated to a set of NaHS standards to obtain a trend line and equation needed to transform the mV readings into H 2 S concentrations. For homogenized extracts in vitro, an equal amount of protein (100-300 μg) was added to a final reaction containing 10mM Cys and 10 μM PLP in a 96-well plate and covered with cellophane to isolate each well and trap any gas that was produced. After 3 hr of incubation, mV readings were taken after inserting the probe through the cellophane covering into each well individually. For in vivo measurements of endogenous H 2 S concentrations, the probe was inserted directly into median and left liver lobes of anesthetized mice prior to sacrifice.

Simulated In Vitro DR Hepa1-6 cells were cultured in complete RPMI-1640 (Sigma) supplemented with 10% FBS at 37°C in 20% O 2 until ∼70%–80% confluence. Equal numbers of cells were then seeded into 96-well format in complete RPMI-1640. When cultures obtained ∼70%–80% confluence, the media was removed and replaced either with complete RPMI-1640 or RPMI lacking Met and Cys (Sigma), both supplemented with 10% dialyzed FBS, and the cells incubated overnight for 16 hr. Primary hepatocytes were isolated by collagenase treatment (Liberase, Roche), Percoll (GE Healthcare) gradient centrifugation and initially cultured in William’s E media (Sigma) with 5% FBS for several hours. The media was then removed and replaced with complete or Met and Cys null DMEM-12320 media (Invitrogen) supplemented with 10% dialyzed FBS overnight for 16 hr. Primary mouse aortic smooth muscle cells were prepared from thoracic aorta. Following dissection of periadventitial tissues under a dissecting microscope, the aorta was minced into small pieces and placed as explants, luminal side down, on the dry surface of a 24-well culture plate previously coated with 7% Cell-Tak (Corning). Explants were gently covered with one drop of RPMI, 10%FBS medium, and placed overnight in a 37°C, 5% CO 2 . The next day, culture medium was carefully added to the wells without detaching the explants. SMCs were identified by immunostaining using antibodies to smooth muscle actin (abcam, ab5694) and desmin (Dako, M 0760). Passages 1 to 4 were used for the experiments. For in vitro DR, complete media was removed and replaced with either complete or Met and Cys null DMEM (Invitrogen) for up to 20hrs prior to H 2 S detection.

Simulated In Vitro Ischemia Reperfusion Injury and Oxidative Stress Growth or preconditioning media was replaced with saline to mimic nutrient/energy deprivation, with or without NaHS (10 μM) or thiosulfate (1, 10, or 100 μM, Sigma). For hypoxia, plates were placed in an airtight chamber flushed with nitrogen gas at 37°C for 4hrs. At the end of the hypoxic period, the saline supernatant was removed and tested for cell damage (LDH release). Reperfusion was simulated by adding back fresh complete DMEM-12320 containing 0.5mg/mL MTT (Invitrogen) and incubating at normoxia at 37°C for an additional 3-4hrs. The media was removed and replaced with acid isopropanol (0.04M HCl in isopropanol) and absorbance read at 520nM or analyzed for LDH activity. H 2 S-mediated protection from oxidative stress in Hepa1-6 or primary vascular smooth muscle cells was performed by adding 10 μM or 100 μM H 2 S to the cell culture media prior to challenge with H 2 O 2 at a final concentration of 5mM for Hepa1-6 and 1mM for smooth muscle cells 24hrs prior to analysis for LDH and MTT.

siRNA Knockdown In Vitro Módis et al., 2013 Módis K.

Coletta C.

Erdélyi K.

Papapetropoulos A.

Szabo C. Intramitochondrial hydrogen sulfide production by 3-mercaptopyruvate sulfurtransferase maintains mitochondrial electron flow and supports cellular bioenergetics. siRNA knockdown of mouse Sulfide Quinone Oxidoreductase (SQR) was performed in Hepa1-6 cells as described previously () with the following siRNA oligos: Sense: GACGAGAACUGUAUCCGCAtt and AntiSense: UGCGGAUACAGUUCUCGUCtg, with RNA in uppercase and DNA in lowercase. Knockdown was confirmed by SQR immunoblot (ProteinTech).

Fly Conditions 2 , sorted by sex and then female flies were transferred to cages on the fully defined medium containing 62.08 g Diet TD.04310 (corresponds to the 0.1X diet) and/or 101.07 g Diet TD.10417 (corresponds to the 1.0X diet) (Harlan Teklad), 100mg lecithin from soybean (Sigma), 500 mg ribonucleic acid from Torula yeast (Sigma), 100 g of dextrose, 20 g agar, 2.85mL propionic acid, 0.255mL of phosphoric acid (Sigma), and indicated amounts of Met (Sigma) per liter of water. To prepare 0.4X and 0.7X diets, the two basal mixes were mixed to adjust amino acid concentrations ( Lee et al., 2014 Lee B.C.

Kaya A.

Ma S.

Kim G.

Gerashchenko M.V.

Yim S.H.

Hu Z.

Harshman L.G.

Gladyshev V.N. Methionine restriction extends lifespan of Drosophila melanogaster under conditions of low amino-acid status. 2 S production assay. D. melanogaster (Canton-S) line was from the laboratory of Dr. Lawrence G. Harshman (University of Nebraska, Lincoln, NE). Flies were maintained on corn meal food and kept in a temperature-controlled chamber at 25°C with 12hr light/dark cycle and approximately 60% humidity. Newly emerged flies were transferred to fresh corn meal food and allowed to mate for 1-2 days. Three day old mated flies were collected using CO, sorted by sex and then female flies were transferred to cages on the fully defined medium containing 62.08 g Diet TD.04310 (corresponds to the 0.1X diet) and/or 101.07 g Diet TD.10417 (corresponds to the 1.0X diet) (Harlan Teklad), 100mg lecithin from soybean (Sigma), 500 mg ribonucleic acid from Torula yeast (Sigma), 100 g of dextrose, 20 g agar, 2.85mL propionic acid, 0.255mL of phosphoric acid (Sigma), and indicated amounts of Met (Sigma) per liter of water. To prepare 0.4X and 0.7X diets, the two basal mixes were mixed to adjust amino acid concentrations (). The experimental flies were held on the designed diet and transferred to fresh vials without anesthesia every three days. After 18 days, all flies were collected and then used for HS production assay.

Worm Conditions Mair et al., 2011 Mair W.

Morantte I.

Rodrigues A.P.

Manning G.

Montminy M.

Shaw R.J.

Dillin A. Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB. N2 and DA1116 (eat-2(ad1116) II.) strains were grown on 6 cm nematode growth media plates following standard procedures. RNAi clones were from Ahringer RNAi library. RNAi bacterial cultures were grown overnight at 37°C in the presence of carbenicillin (100 μg/mL) and tetracycline (10 μg/mL) before seeding to NGM plates containing carbenicillin (100 μg/mL). Seeded RNAi plates were allowed to grow at room temperature for 48 hr. Expression of dsRNA was induced with 100 μl of IPTG (100 mM) 2 hr before worms were transferred to the plates. All lifespan experiments were carried out as previously described (). In summary, worms were synchronized with a timed egg lay or bleach. Eggs were allowed to hatch and grow to adulthood on control or RNAi plates, respectively. Day 1 of lifespan assay was defined as the first day of adulthood. Worms were transferred to fresh RNAi plates and scored for death or censor events every 1-2 days until day 14, after which they were scored every 1-2 days but only transferred once per week. Death was scored as no response to gentle agitation at the head or tail. Those worms that died as a result of internal hatching or crawling off the plates were scored as censored. Transgenic C. elegans strain construction for CBS overexpression: C. elegans cbs-1 isoform a (ZC373.1a) was PCR amplified and subsequently fused in-frame to the N terminus of tandem-dimer TOMATO. The fusion protein was expressed using the ubiquitous sur-5 promoter and unc-54 3′ UTR. 50 ng/μl of the cbs-1 construct was mixed with 50 ng/μl of pCFJ104 (myo-3p:mCherry) as co-injection marker and microinjected into gonads of hermaphrodites to generate transgenic strains.

Yeast Conditions Experiments were carried out in wild-type strains BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), W303 (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15), and DBY746 (MATα leu2-3, 112 his3Δ1 trp1-289 ura 3-52 GAL+) grown in SCD media. Met deletion strains in BY4742 (met5, met14 and met16) were purchased from EUROSCARF. Composition of SCD media: 0.17% yeast nitrogen base (BD Diagnostics; without ammonium sulfate and amino acids), 0.5% (NH 4 ) 2 SO 4 , 30mg/L of all amino acids (except 80mg/L histidine and 200mg/L leucine), 30mg/L adenine, and 320mg/L uracil with the indicated amounts of glucose (0.5% for DR, and 2% for standard conditions, respectively). All amino acids were purchased from Serva (research grade, ≥ 98.5%). For chronological aging experiments, cells were inoculated to an OD600 of 0.05, and grown at 28°C in SCD media. At the indicated time points, cell survival was determined by clonogenicity: Cell cultures were counted with a CASY cell counter (Schärfe System) and 500 cells were plated on YPD agar plates (at least 3 independent cultures were evaluated) and subsequently colony forming units counted and values normalized to survival at day one. For chronological aging experiments with externally added H2S-releasing substances, GYY4137 (Sigma) dissolved in DMSO was added to BY4742 cultures at time of inoculation at a final concentration of 100μM. In addition, NaHS (Sigma) was added at time points 6, 24 and 48hrs after inoculation at concentrations of 5 μM at each time point. DMSO-treated BY4742 strain served as control.