C www.landesbioscience.com sues and solid tumors have been well-characterized. been have tumors solid and sues tis normal in to hypoxia response adaptive and sensing Oxygen nutrient environments. dynamic to the proliferation and survival adaptation, their determines oxygen and source nitrogen source, of carbon lack properly to transient respond and to sense cells of local ability the or tumors, solid tissues ischemic In comes. out clinical deleterious and acids amino and glucose oxygen, of lack to the leads disorders, some cardiovascular and tumors solid in occurring condition pathological acommon Ischemia, of tumor progression. tumor of hallmark emerging an as regarded been has metabolism energy of Reprogramming oxygen. molecular and sources nitrogen of Glc or Gln. to availability respond and sense how cells unclear providing of ATP, reducing power and building blocks. of ATP,providing building and power reducing through proliferation and function cell supporting in roles cal playcriti oxygen molecular and source nitrogen source, Carbon to be indispensible for most cells in in vitro culture systems. culture vitro in in cells for most indispensible to be found been have (Gln) glutamine and (Glc) glucose particular, 1 Department of Biology; College of Arts and Sciences; Drexel University; University; Drexel Sciences; and Arts of College Biology; of Department http://dx.doi.org/10.4161/cc.21944 02/09/12;Submitted: 08/21/12; Revised: Accepted: 08/23/12 to: Nianli*Correspondence Email: Sang; [email protected] ell Cycle 1,2012;©2012LandesBioscience 11:19,3679-3690;October of Medicine; Medicine; of Particularly, tumor cells require increased supply of carbon, supply of carbon, increased require cells tumor Particularly, Shuo Qie, Shuo Rep trigger growth gene inhibitionviadistinctive or Glutamine depletionandglucoseGlutamine depletion the biological behaviors of cells. of behaviors biological the on abundance Gln and Glc of effects potential and mechanisms nutrient-sensing the investigating further for resource a provides database profiling mRNA Our insufficiency. Glc and Gln to respond and sense to mechanisms different use cells and proliferation, and cellsurvival supporting in roles metabolic have distinctive Glc and thatGln support patterns expression profiles. Moreover, Gln and Glc depletion triggered distinguishable distinguishable triggered depletion Glc Moreover, and Gln profiles. expression gene and regulators transcription on effects have conditions distinctive these but genes, cell cycle of expression the of reprogramming. expression gene dramatic triggered depletion Glc and Gln that revealed data profiling mRNA of Analysis conditions. nutrient defined the under cells cultured those in expression gene the of changes global the we report Here depletion. to Gln cells cells adapted and Glc-depleted cells, Gln-depleted acute of profiles expression gene the wecompared techniques, unclear. profiling remains mRNA Using insufficiency Glc and to Gln respond and sense cells how directly well-studied, have to been hypoxia mechanisms adaptive and sensing oxygen- Whereas insufficiency. nutrient and hypoxia with is associated which supply, ischemia, in resulting blood the outgrow often tumors Solid celldeath. eventually and arrest growth to trigger shown have been depletion Glc and depletion Gln glutaminolysis. and glycolysis enhanced with associated are processes thatoncogenic observations the consistent with cells, proliferating for nutrients important (Gln)two (Glc) glucose represent and Glutamine t P h iladelphia, iladelphia, 1,2 Dongming Liang, P Keywords: hi ladelphia, ladelphia, 6 P As consequences of oncogenic signaling signaling of oncogenic consequences As A U

Introduction SA; SA; P 3 A glutamine depletion, glucose depletion, metabolism, ER stress, ATF4, XBP1 ATF6, stress, ER depletion, metabolism, depletion, glucose glutamine Departments of Cancer Biology; Stem Cell Biology and Regenerative Medicine; Kimmel Cancer Center; Center; Cancer Kimmel Medicine; Regenerative and Biology Cell Stem Biology; Cancer of Departments expression reprogramming USA; 4 Department ofDepartment Obstetrics and Gynecology; Qilu Hospital; Shandong University; Jinan, Shandong, China 1 Chengqian Yin, Chengqian 4,5 It remains It remains 1 Weiting Gu, P h iladelphia, iladelphia, 1,2 C In In ell Cycle 2,3 - - -

P A U glutamate catalyzed by glutaminase. catalyzed glutamate synthesis. acid fatty as such activity, biosynthetic for power increased reducing and carbons anabolic with cells tumor proliferative provides Warburg effect tion. for prolifera glutaminolysis to active addicted become also cells brane mem plasma to the transporters of glucose translocation and/or expression the as such reprogramming, of metabolic to aseries lead that genes suppressor of tumor loss and stimulation genic ferent regions of tumors. To survive in such stressful conditions, conditions, stressful such To in of tumors. ferent regions survive dif in nutrient distribution uneven and ischemia to localized ing termed the Warburg effect. the termed aphenomenon aerobic conditions, in even cells, bytion tumor phosphoryla of oxidative ATP instead to generate glycolysis via metabolized is glucose source, a major carbon As radiation. and to chemotherapy for resistance basis biochemical the form which features, metabolic show aberrant cells tumor adaptation, and reductive precursor for highly proliferative tumor cells. tumor proliferative for highly precursor reductive and biosynthetic energetic, an as used be can and nitrogen and 3,4

SA; SA; Meng Meng, Meng Solid tumors usually have heterogeneous blood supply, blood heterogeneous have lead usually tumors Solid 2 14,15 Department of of Department 9,10 Glutaminolysis defines the conversion of glutamine to conversiontheglutamine of defines Glutaminolysis and upregulation or activation of glycolytic enzymes. of glycolytic or activation upregulation and E i 1 ther Gln or Glc deletion leads to changes to changes leads deletion Glc or Gln ther E P Chenguang Wang a R thology and Laboratory Medicine; Drexel University College College University Drexel Medicine; Laboratory and thology -stress responses. responses. -stress 7,8 12,13 Warburg effect is the result of onco result the is Warburg effect Besides Warburg effect, tumor tumor Warburg effect, Besides T 14 3,4 h Gln contains both carbon carbon both contains Gln and Nianli Sang Nianli and e gene expression expression e gene T h omas Jefferson University; University; Jefferson omas 16,17 1,2, r * e Por 3679 11 t ------

©2012 Landes Bioscience. Do not distribute. 3680 responding molecule to hypoxia. to molecule responding best-known the is (HIF) inducible factor Hypoxia ming. reprogram metabolic and to transcriptional resort cells tumor damage repair and so forth. so and repair damage DNA progression, cycle cell synthetase, aminoacyl-tRNA ers, transport family solute carrier regulators, transcriptional other including (AAREs), elements response acid amino with of genes (eIF2 2 factor initiation eukaryotic phosphorylates which GCN2, activates GCN2 with tRNAs of uncharged binding direct The (IRS). response stress integrated termed response, stress aconserved in results or leucine, cysteine as such acids, of amino Deprivation plex ( plex com of ternary formation the by inhibiting translation global (GCN2) senses the abundance of non-aminoacylated tRNAs. of non-aminoacylated abundance the senses (GCN2) relevant genes that induce the adaptive response to hypoxia. response adaptive the induce that genes relevant other and glycolytic of angiogenic, expression the upregulates proliferation. Starting from Hep3B, we have established a cell acell Hep3B, from established we have Starting proliferation. for Gln on Glc and depends hepatocytes, from derived line cell to Glc depletion. response in cells tumor in expression of gene analysis no systemic is there cells, forpensible most proven indis to been be Glc has while Similarly, depletion. Gln to response how cells unclear However, source. itgen remains anitro and source acarbon both as playroles may Gln systems. culture vitro in in cells for most essential to be shown been has (ATF4). 4 factor transcription activating as such mRNAs, of selected 0.001). (p < significant statistically were presented data All indicated. were deviations Figure 1. sample and tested gene, qR gene, tested and sample each For shown. were cells Gln-depleted in mRNA of levels as Relative 1. defined arbitrarily were control the in levels mRNA and control, as used were media plete determined by qR by determined profiling. mRNA by identified genes representative of upregulation the of (B) Validation MM01 cells. and Hep3B Glc-depleted or Gln- in pattern expression gene of map We have showed that the proliferation of Hep3B, proliferation We atumor the that showed have

α Met-tRNA 19 ). The phosphorylation of eIF2 phosphorylation The ). During IRS, general control nonderepressible kinase 2 controlnonderepressible kinase general IRS, During 21-23 Gene expression in Hep3B cells with different treatments. (A) Heat- (A) treatments. different with cells Hep3B in expression Gene T a qMan primers were purchased from Invitrogen. Levels of mRNA were were mRNA of Levels Invitrogen. from purchased were primers qMan Increased ATF4 translation leads to the expression expression to the leads ATF4 translation Increased i Met T - P •eIF2-GTP), while increasing the translation translation the increasing while •eIF2-GTP), C R. RNA samples isolated from Hep3B cells cultured in com- in cultured cells Hep3B from isolated samples RNA R. T - P C 24-27 R was performed at triplicate, and standard standard and triplicate, at performed R was Gln, a nonessential amino acid, acid, amino a nonessential Gln, 4 Under hypoxic condition, HIF HIF condition, Under hypoxic results in decreased decreased in α results C ell Cycle α 20 18 - - - - -

tion were downregulated by Glc( depletion were downregulated tion ( depletion Gln by by Glc were downregulated depletion upregulated genes 68 ( depletion Gln Glc and depletion by both ( portion small avery represent depletion, 71 ortion Gln genes overlapped depletion ( depletion by Glc regulated those with depletion by Gln regulated genes the compared we next of group genes, same of the regulation the to lead and to cells stresses similar Glc cause depletion and tion upregulated. The upregulated genes with alog with genes upregulated The upregulated. selected genes upregulated at different levels (log levels atdifferent upregulated genes selected 1 Table in listed function and metabolism. and function cell in normal roles Gln of important the underlining profiling, of expression gene change amuch greater caused apparently tion deple to Glc depletion, Gln We comparing lated. noted that were downregu genes 662 while 590 were upregulated, them, tial expression of 1,252 expression (log tial genes by qRT-PCR. confirmed be can of regulation) (direction 1B Figure in shown Results PCR. qRT- using levels mRNA their examined and 1.5–4.7) from genes (log genes of 2,197 expression differential in (for 24resulted h) depletion S1 Table in were summarized 1A, Figure in shown was of expression gene heatmap The microarrays. with condition for each triplicate in performed was profiling expression for Gene 24 h. media free

On the other hand, Glc depletion (for 24 h) causes differen Glc (for 24causes h) depletion hand, other On the but not death of Hep3Bbut not death cells. arrest growth in or Glcofdepletion Gln resulted either terns. pat expression gene alters or depletion Glc Gln Either of cells. behaviors biological on the abundance Gln and of Glc effects the and nutrient-sensingmechanisms the of understanding thorough the toward research future facilitate will results Our factors. of transcription ent sets differ by regulating expression gene distinctive triggered Gln- orGlc and found depletion data, profiling mRNA of the analysis functional (IPA), we did analysis way path ingenuity Using MM01 cells. of adapted that and media Glc-depleted in cultured of cells that with media Gln-depleted in of profile Hep3Bcells expression mRNA genome-wide the compared and examined systemically in complete media (control), Gln-free media or Glc- media Gln-free (control), complete media in cultured Hep3B from cells samples RNA total isolated pathways for anabolism of carbon and nitrogen. and of carbon for anabolism pathways metabolic alternative utilize to adaptively ability the has to study, amodel for as it hepatocytes, from originated line cell chose Hep3B, profiling. We tumor a expression Glcby gene depletion and Gln depletion, we compared Gln to the responses of cellular understanding the line, MM01, which has adapted to Gln-free media. to Gln-free adapted MM01,line, has which Fig. 2A Fig. 2 Ratio > 2.0 or < -2.0); among them 638 genes were 638 genes them among or <-2.0); >2.0 Ratio Fig. 2 Fig. We showed previously that short-term (24–48 h) short-term (24–48 We that previously showed Fig. 2C . To validate the results from gene profiling, we profiling, gene from . To results the validate ). Similarly, only 133 genes were downregulated 133 only Similarly, were downregulated ). genes ). Among the genes upregulated by Glc deple upregulated genes the Among ). ), and 17 genes upregulated by Gln deple by Gln 17 and ), upregulated genes 3 To understand whether Gln deple To Gln whether understand . Compared with control cells, Gln Gln controlcells, with . Compared Results 2 indicate that the upregulation upregulation the that indicate Ratio >1.0 or <-1.0); Ratio among 14 As the first step toward toward step first the As Fig. 2B Fig. Fig. 2D Fig. and detected genes genes detected and 2 V Ratio > 3.0 are >3.0 are Ratio olume 11 Issue19 ). Interestingly, Interestingly, ). 2 values range range values ). Since both both Since ). 14 14 We We We We ------

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com Genes most upregulated by Gln depletion (log depletion Gln by upregulated most Table 1. Genes Rank 40 25 34 28 30 29 83.074656 38 36 26 23 22 27 20 39 35 33 42 32 24 37 10 16 21 18 31 43.457144 14 41 19 15 13 12 17 11 6 5 4 1 7 3 2 8 9 3.444888 3.206867 3.268685 3.373006 3.825807 3.304534 3.223594 3.246922 3.260832 3.200373 3.059862 3.088076 3.378495 3.897732 3.061844 3.220771 3.586737 3.541464 3.496547 3.201466 4.712066 3.343561 3.256531 .3892.27 3.337839 3.070325 3.254313 3.265561 3.057593 3.828137 3.647815 3.661515 4.116244 3.159755 3.127714 3.159169 3.117394 3.114287 4.04208 3.68936 3.88152 Log2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 8.55 1.34 1.40 3.86 1.40 3.82 5.63 1.57 1.57 1.52 2.21 7.43 p value

E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ------2 - - - -3 -2 37 39 35 31 39 44 40 45 45 42 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 9 9 4 MEF2B|MEF2B OSTBETA GADD45G MAPK8IP2 TMEM198 ANKRD11 FAM134A PLEKHA4 TSC22D3 PPP1R3G SCN2B ZSWIM4 FAM65C TBC1D2 CYP2B6 ZNF395 KLHL29 TRIOBP SLC1A4 SHMT2 UBOX5 PI4KB RASIP1 Genes FOXQ1 WWC1 SPIRE1 FOXP4 GPR56 PACS2 BATF2 MBD6 TIGD2 S100P POP4 GPT2 HEY1 G0S2 GEFT ETV5 STC2 LRP3 IDS mitogen-activated protein kinase 8 interacting protein 2 protein 8interacting kinase protein mitogen-activated protein phosphatase 1, regulatory (inhibitor) subunit 3G subunit (inhibitor) 1, regulatory phosphatase protein processing of precursor 4, ribonuclease ribonuclease 4, precursor of processing cytochrome cytochrome 2 solute carrier family 1 (glutamate/neutral amino acid acid amino 1(glutamate/neutral family carrier solute Ratio >3) Ratio growth arrest and DNA-damage-inducible, gamma and DNA-damage-inducible, arrest growth serine hydroxymethyltransferase 2 (mitochondrial) M basic leucine zipper transcription factor, A factor, transcription zipper leucine basic low density lipoprotein receptor-related protein 3 protein receptor-related lipoprotein density low pleckstrin homology domain containing, family A family containing, domain homology pleckstrin hairy/enhancer-of-split related with YR with related hairy/enhancer-of-split family with sequence similarity 134, similarity withsequence A family member E (phosphoinositide binding specific) member4 family with sequence similarity 65, C similarity member withsequence family phosphofurin acidic cluster sorting protein 2 protein sorting cluster acidic phosphofurin F phosphatidylinositol 4-kinase, catalytic, β catalytic, 4-kinase, phosphatidylinositol sodium channel, voltage-gated, type II, II, type voltage-gated, channel, sodium 2B read through transcript |myocyte enhancer enhancer |myocyte transcript through 2B read methyl-CpG binding domain protein domain 6 binding methyl-CpG tigger transposable element derived 2 zinc containing finger, 4 SWIM-type RhoA/RAC/CDC42 exchange factor exchange RhoA/RAC/CDC42 WW and C2 domain containing 1 containing domain C2 and WW T T glutamic pyruvate transaminase T S100 protein calciumbinding G protein-coupled receptor 56 receptor G protein-coupled R S organic β solute transporter spire homolog 1 (Drosophila) P B (alanine 2 aminotransferase) transmembrane protein 198 protein transmembrane IO and F-actin binding protein binding and F-actin IO C22 domain family, member 3 member family, domain C22 U-box domain containing 5 containing domain U-box 4 C1 domain family, member 2 member family, C1 domain ankyrin repeat domain 11 domain repeat ankyrin kelch-like 29 (Drosophila) kelch-like Ras interacting protein 1 protein interacting Ras C 50, family 2, subfamily B, polypeptide 6 polypeptide B, subfamily 2, 50, family transporter), member 4 member transporter), zinc finger protein 395 protein finger zinc ell Cycle iduronate 2-sulfatase iduronate forkhead box Q1 box forkhead forkhead box box forkhead stanniocalcin 2 stanniocalcin G ( ets variant 5 variant ets S. cerevisiae S. 0 /G1switch 2 /G1switch factor 2B factor Names

) P 4 P /M P P W m R T P F subunit subunit -like 2 -like otif 1 otif β M_0157.,M_001145772.1 _001145774.1,NM NM M_0182.,M_001128626.1 _001128627.1,NM NM N NM_001161662.1,NM_001161661.1 NM_001040708.1,NM_012258.3 NM_000202.5,NM_001166550.1 NM_001039141.2,NM_007032.5 NM_020904.2,NM_001161354.1 NM_001166356.1,NM_005412.5 NM_001135581.1,NM_003038.3 NM_001142466.1,NM_133443.2 M_001012426.1,NM_001012427.1 NM_001015881.1,NM_198057.2 NM_015197.2,NM_001100913.1 M_8972N _001111270.1 _182947.2,NM NM NM_016431.3,NM_012324.3 NM_014948.2,NM_199415.1 NR_027368.1,NM_006627.2 NM_000767.4|NR_001278.1 NR_027308.1,NR_027307.1 NM_001005209.1 Access Numbers M_001145115.1 NM NM_004588.4 NM_178859.3 NM_002651.1 NM_004454.2 NM NM_080829.2 NM_006705.3 NM_002333.3 NM_018660.2 NM_023072.2 NM_033260.3 NM_003714.2 NM_138456.3 NM_017805.2 NM_024293.4 NM_052920.1 NM_052897.3 NM_018421.3 NM_015714.3 NM_013275.4 NM_145715.2 _005980.2 3681

©2012 Landes Bioscience. Do not distribute. 3682 changes in genes controlling the G the controlling genes in changes more significant However, caused depletion Gln changed. cantly were signifi regulators cycle cell other and of cyclins patterns orexpression depletion Glc depletion, the Gln either under that Gln or Glc depletion increased cell population in G in population cell or Glcincreased depletion Gln either found and analysis cycle cell we performed progression, Figure 2E Figure in shown Data regulators. cycle of cell groups of several expression the analyzed we particularly inhibition, growth similar Glc cause depletion and depletion Gln G involved in of genes expression the affected primarily depletion depletion. ways. different in respond but cells stresses, depletion nutrient Glc represent depletion and depletion Gln both that indicate changes cycle cell and patterns expression gene distinct ( Fig. S1 Fig. Genes most upregulated by Gln depletion (log depletion Gln by upregulated most Table 1. Genes Glc depletion mainly caused the reprogramming of genes regulating G regulating genes of reprogramming the caused mainly depletion Glc was significantly changed. In particular, Gln depletion significantly altered the expression profiling of genes regulating G regulating genes of profiling expression the altered significantly depletion Gln particular, In changed. significantly was regulators cycle cell and cyclins of expression the conditions, nutrient both under that show data Our x-axis. as -log in presented and algorithm, and I by analyzed was expression ( conditions. both under downregulated or conditions, both under regulated expression levels, log For Glc depletion, log Figure 2. Rank 1 /S. To understand if these changes were related to cell cycle cycle to cell were related changes To these if /S. understand 44 46 48 50 45 49 43 47 51 Functional responses of transcription regulators to Gln Gln to regulators of transcription responses Functional

), which is consistent with IPA ( with prediction consistent is which ), 3.054969 3.005587 3.032083 3.025387 3.045242 3.012984 3.015883 Gln depletion and Glc depletion alter gene expression. (A–D) RNA samples from Hep3B cultured in complete media were used as control. control. as used were media complete in cultured Hep3B from samples RNA (A–D) expression. gene alter depletion Glc and depletion Gln 3.00214 3.01661 It has been well known that amino acid starvation starvation acid amino that known well been It has Log2 2 2 Ratio > 2.0 (upregulation) or < -2.0 (downregulation) was set as cut-off. We noted that only a small portion of genes were up- were genes of portion small a only that noted We cut-off. as set was <-2.0 or (downregulation) (upregulation) >2.0 Ratio 0.00 0.00 0.00 Ratio > 1 (upregulation) or < -1.0 (downregulation) was used as cut-off. Since Gln depletion caused dramatic change of gene gene of change dramatic caused depletion Gln Since cut-off. as <-1.0 used or was >1(upregulation) (downregulation) Ratio 4.82 4.42 9.42 1.10 1.41 7.71 p value E E E E E E E E E + + + ------26 27 32 31 33 P 41 00 00 00 A . P value of the reprogramming of each group of cell cycle regulators were calculated according to established criteria criteria toestablished according calculated were regulators cycle cell of group each of reprogramming the of value WHSC1L1 C1orf216 KCNMB3 FBXO31 Genes FOXC1 2 PLA1A TCEB2 FBLL1 PELI3 /M checkpoint, whereas Glc whereas checkpoint, /M and Table and S2 potassium large conductance calcium-activated channel, channel, calcium-activated large conductance potassium 2 transcription elongation factor B (SIII), polypeptide 2 B(SIII), polypeptide factor elongation transcription Ratio >3) Ratio Fig. 2E Fig. Wolf-Hirschhorn syndrome candidate 1-like1 candidate syndrome Wolf-Hirschhorn 2 chromosome 1 open reading frame 216 frame reading 1open chromosome /M phase phase /M indicate indicate pellino homolog 3 (Drosophila) (continuted) ). The The ). phospholipase A1 member A subfamily Mβmember3 subfamily C ell Cycle 1 /S checkpoint. (18kDa, elongin B) (18kDa, elongin - F-box protein 31 protein F-box forkhead box C1 box forkhead fibrillarin-like 1 fibrillarin-like E of ATF4 target genes. of ATF4 target upregulation the in resulting ATF4 translation, IRES-initiated stimulates but of specifically proteins, translation the inhibits of GCN2-eIF2 activation the triggers parameter to estimate confidence level. Based on this analysis, 25 analysis, this on Based level. confidence to estimate parameter objective an as z-score the uses and genes, targeted downstream known of all response on the based regulator transcription given of a z-scores regulation calculates IPA the program criteria, and algorithms established Using genes. downstream known of their (up or downregulated) directions expression on the based lators regu of transcription or inhibition activation functional the dicts pre which IPA the program, with set data expression gene our We analyzed status. functional of their indicators reliable most not the are regulators of transcription levels mRNA alterations, or pathological of physiological by avariety regulated tors are regula oftranscription most functions the Because implicated. were regulators transcription other we wondered if expression, Names )

E x pression of cell cycle regulators in Gln- and Glc-depleted cells. Gene Gene cells. Glc-depleted and Gln- in regulators cycle cell of pression 26 Considering the breadth of altered gene gene of altered breadth the Considering NM_001098510.1,NM_145065.2 NM_207013.1,NM_007108.2 NM_171830.1,NM_014407.3 NM_024735.3,NR_024568.1 2 /M and G /M and axis, which generally generally which α axis, Access Numbers NM_015900.2 NM_001453.2 NM_017778.2 NM_152374.1 NR_024356.2 1 /S checkpoints, and and /S checkpoints, V olume 11 Issue19 - - -

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com transcription complexes. transcription componentas a of bHLHb21,multiple identified and been has growth. TOB1 cell cells, suppresses cultured in expressed exogenously when that reported been It proteins. has of anti-proliferative ily fam tob/btg1 of amember the encodes of ERBB2) (transducer ming of G ming reprogram the with consistent is observation This arrest. growth p53, and depletion-triggered Gln pRb involved in are regulators, cycle cell pivotal two the that p53 suggesting stability, regulating aprotein encodes which MDM2, and RB, repressor cycle cell ( >2.00) z-score (regulation activated to be were predicted lators regu transcription eight of function the that to find were puzzled depletion, we upon Gln were repressed regulators transcription many that to observe not surprising it was Although derivatives. metabolic its and Gln to conserve repressed generally are tocytes of hepa functions depletion, normal upon Gln that indicating FOXO1, and SREBF2 HNF1, are HNF4, regulators scription tran inhibited putatively these Among inhibited. to be predicted were -2.00, thus, than smaller were scored regulators transcription to repress the transcription of a variety of genes. of avariety transcription the to repress 1, corepressor receptor nuclear 1and Sirtuin as such complexes, deacetylase histone with interacts and homo- or hetero-dimers either protein forms encoded The regulator. transcription type (bHLH)- helix-loop-helix of basic family of split-related (HESR) represses Myc-dependent transcription. MAD HDAC2 Sin3A, and recruits also MAD Since erodimers. het to form Myc for Max with competes protein, which MAD pressor gene in uterine leiomyomas and breast cancers. breast and leiomyomas uterine in gene pressor sup atumor as considered been has It also atS-phase. ticularly par progression, cycle cell and differentiation morphogenesis, to regulate found been has protein and Rb-interacting an is Cux) ( depletion upon Gln frequently in pleomorphic adenomas of the salivary glands. salivary of the pleomorphic adenomas in frequently observed of PLAG1been has translocation Chromosomal lated. regu developmentally is expression and its protein, finger zinc PLAG1 a 1) gene adenoma (pleomorphic Particularly, encodes before. reported not been involvement nutrient has in depletion their and regulators, transcription activated as were scored be inhibited ( inhibited be and N-Myc of to found Myc were functions the finding, this tion ( tion of PLAG1 deple by activation Gln the predicts IPA analysis factor 1(CHF1). factor helix-loop-helix cardiovascular as known also is protein 2) motif nitrogen source for the synthesis of nitrogenous biomolecues. of nitrogenous synthesis for the source nitrogen ( depletion of Gln that from distinct clearly by Glcis depletion triggered of expression gene However, alteration the biosynthesis. liferative for pro sources carbon role as metabolic acommon share both depletion. determined. to be remains depletion upon Gln regulators transcription of these activation Table 2 Table In addition, PLAG1, addition, In TCF3, TOB1, MXD1 and Cux1, HEY2 Functional responses of transcription regulators to Glc Glc to regulators of transcription responses Functional Table 3 Table 31 ). Among these activated regulators are the renowned renowned the are regulators activated these Among ). HEY2 (hairy/enhancer-of-split related with YRPW YRPW with related (hairy/enhancer-of-split HEY2 Both Glc and Gln are indispensible for most cells, and and cells, for most indispensible are Gln Glc and Both 1 Fig. 1A and 2 and Fig. 1A /S and G and /S Table 1 Table ). TCF3, also known as E2A, E47, E2A, as ITF1, VDIR known TCF3,). also 32 HEY2 is a member of the hairy and enhancer enhancer and hairy of amember the is HEY2 2 /M checkpoint regulators ( regulators checkpoint /M ). The precise biological significance of the of significance biological precise The ). Table S3 Table

29 ). In addition, Gln is also an important important an also is Gln addition, In ). IPA analysis suggests TCF3 activation TCF3 activation suggests IPA analysis ). Cux1 (CUTL1, CDP, (CUTL1, Cux1 ). CDP/ 34,35 Consistent with with Consistent 33 Fig. 2E Fig. MXD1 encodes encodes MXD1 ). 30 TOB1 TOB1 14 C To To ell Cycle 28 ------

vated regulators, consistent with reports from other independent independent other from reports with consistent regulators, vated acti these among were found responses, ER-stress involved in depletion. by Gln triggered that from different by Glcare depletion caused stresses cellular the that gest sug findings These hepatocytes. in regulation metabolic normal in playroles regulators transcription inhibited functionally Other by Glc depletion. repressed regulators the among found are E2F2 E2F1 and both addition, In Glc depletion. with associated arrest growth cell observed the with consistent suppressors, liferative pro are TP63 and RB1, TP73, YY1 CDKN2A, activated The levels). at functional activated to be predicted being (thus 2.0 in shown As data. microarray the IPAdepletion, we used to analyze to Glc response involved in regulators transcription potential tify iden and regulators of transcription states functional the predict depletion toGln regulators transcription of responses Functional Table 2. were listed for reference. *

T Table 4 Table r Notably, ATF4 and ATF6, two regulators known to be to be known Notably, regulators two ATF6, ATF4 and Transcription anscription regulators with z-scores between 1.65–2.0, -1.65–-2.0 or between z-scores with regulators anscription Regulator T NKX2–1 Cyclin NF KDM5B MYCB N4 niie 2626.92 -2.662 Inhibited HNF4A N1 niie 3162.53 -3.116 Inhibited HNF1A OO niie 2701.45 -2.780 Inhibited FOXO1 D2Atvtd2.146 Activated MDM2 c P T X1Atvtd2.016 Activated MXD1 YNIhbtd-.4 2.65 -3.440 Inhibited MYCN HIF1A U1Atvtd2.217 Activated CUX1 R7Ihbtd-.5 3.01 -2.254 Inhibited BRD7 T H R1Ihbtd-.6 1.07 -2.261 Inhibited NRF1 E XB T Creb E MYC f 1/3/4 GFI1 P FD A1Atvtd2.685 Activated LAG1 B A OB1 R niie 3086.99 -3.028 Inhibited GR1 Rb F A CF3 F niie 2707.48 -2.750 Inhibited LF4 E E GR P 2A Y2 L niie 3553.67 -3.565 Inhibited 2L2 P , 16 transcription regulators have z-scores greater than than greater z-scores have regulators , 16 transcription 3.48 -2.562 Inhibited 1 niie 2087.70 -2.028 Inhibited 1 P E Predicted ciae 2.041 Activated niie 2091.70 -2.049 Inhibited I niie 2267.64 -2.236 Inhibited niie 2741.00 -2.744 Inhibited hbtd-.4 7.60 -2.049 nhibited tvtd2.076 ctivated tvtd2.514 2.633 ctivated ctivated State Regulation z-score -1.666 -1.843 -1.788 - 1.803 1.696 1.718 p value of 4.30 8.73 1.90 1.53 4.67 8.51 1.08 5.18 5.17 5.02 1.54 2.67 4.21 2.12 overlap E E E E E E E E E E E E E E E E E E E E E E E E E E E E E ------02 -03 -01 -01 -01 -02 -01 -02 -02 -02 -03 -02 -02 -02 -10 -08 -60 -02 -02 -02 -05 -04 -04 - 02 06 02 03 09 02 3683 - - - -

©2012 Landes Bioscience. Do not distribute. 3684 groups of cell cycle-related genes: G genes: cycle-related of cell groups three of reprogramming significant caused depletion Gln acute on IPA analysis, Based regulators. cycle of cell expression gene the depletion, we compared Gln upon acute inhibition growth ( conditions Gln-depleted acute under were downregulated 43 while upregulated, 166 were also genes, upregulated the Among MM01 in cells. downregulated were 398 genes while were upregulated, 292 only genes reveal ysis anal- Microarray mechanisms. adaptive into the insight to reveal we expected Hep3B cells, Gln-depeleted acute with of MM01 cells analysis by IPA predicted the responses of several transcription transcription of several responses by the IPA predicted analysis 3C by qRT-PCR,in Figure confirmed shown further was genes but slowly. of selected Upregulation continually proliferate why MM01 cells explain may results above The altered. cantly were not ofsignifi genes groups these MM01 cells, adapted the ( regulators cycle cell and cyclins point and depletion (z-score: 4.12) ( (z-score: depletion Glcby activated significantly depletion, it by Gln was inhibited studies. adaptation to Gln insufficiency. to Gln adaptation of long-term aresult represents which phenotype, slow growing a MM01 line with acell we established ammonia, mented with depletion. Gln-depletion of directions Table 3. Regulation sion. * P SMARCD3 L Altered gene expression in cells adapted to long-term Gln Gln long-term to adapted cells in expression gene Altered ACVR2B COL9A3 CNKSR1 C11orf9 PTP4A3 S100A2 Genes AG1 function predicted based on directions of target gene expres- gene target of directions on based predicted AG1 function VEGFA KCNJ4 MLXIP

ROM1 TGM2 MAP2 FLNC ETV4 GJB1 PLEC CLTB ATF5 PIGF PPIC 36,37 By culturing Hep3B cells in Gln-free media supple media Gln-free in Hep3B cells By culturing Although XBP1 was predicted to be functionally functionally to be predicted was XBP1 Although Log -5.620 -2.381 -2.954 -1.283 -1.069 -1.529 -1.218 2.006 2.668 2.302 2.082 1.868 2.472 1.297 1.070 2.146 1.435 2.186 1.810 1.515 1.194 2 Ratio Table S4 Table P Downregulates (1)Downregulates (1)Downregulates Downregulates (1)Downregulates L Upregulates (2) Upregulates Upregulates (2) Upregulates Upregulates (2) Upregulates 14 Upregulates (1)Upregulates Upregulates (1)Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates Upregulates (1)Upregulates (1)Upregulates Fig. 3A Fig. AG1 target genes upon upon AG1 genes target Comparing the gene expression expression gene the Comparing idnsPrediction* Findings ). 1 /S checkpoint, G checkpoint, /S ). To gain insight into the into the To). insight gain Fig. 3B Fig. Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Inhibited Inhibited Inhibited Inhibited ). While in in While ). 2 /M check /M . Data . Data C ell Cycle - - - PERK-eIF2 by mediated be can which responses, stress ER triggers depletion conditions. nutrient ( MM01 cells of PPAR activation the suggests IPA analysis intriguing. PPAR and ESR1 SMAD3, ( depletion Gln acute during to proliferate failing cells with HIC1.and Comparing DDIT3, TP73 ESR1, PPARA, SMAD3, SMAD2, include lated upregu to be predicted regulators transcription other ATF4, the 5 Table in summarized are which regulators, Functional responses of transcription regulators toGlc(-) regulators transcription of responses Functional Table 4. NF

Transcription The status of ER-stress signaling pathways in different different in pathways signaling of ER-stress status The k Regulator MRA ciae .1 4.36 2.417 Activated SMARCA4 DNAAtvtd3119.50 3.131 Activated CDKN2A B (complex) MLXI NF D5 ciae .1 1.53 3.811 Activated KDM5B OM niie 2655.99 -2.655 Inhibited FOXM1 HDAC2 DC ciae .7 1.59 2.279 Activated HDAC1 UX niie 2022.61 -2.072 Inhibited RUNX2 N1 niie 2154.45 -2.175 Inhibited HNF1A NR4A2 BRCA1 DDI NRI S M B XB Hdac T TP TP T A A E E E RB GLI2 GLI1 IRF4 YY1 RB1 T BX2 CF3 T 2F1 2F2 2 T T E E A G2 63 73 F4 F6 F4 P L ciae .2 3.77 2.028 Activated 2L2 D1 P P T T 1 P J 4 1 ciae .5 4.12 2.257 Activated 3 L α or GCN2-eIF2 Table S5 Table Predicted A ciae .3 4.28 2.233 Activated ciae .3 1.71 2.437 Activated ciae .0 1.56 A 2.600 Activated ciae .9 6.37 2.295 3.35 Activated 2.685 Activated ciae .1 2.61 4.014 Activated ciae .1 3.16 4.115 Activated It has been well-established that nutrient that well-established been It has niie 2104.42 -2.130 Inhibited niie 2038.84 -2.023 Inhibited niie 2273.70 -2.287 Inhibited niie 3934.47 1.52 -3.943 -2.424 Inhibited Inhibited Table 2 Table tvtd2241.12 2.224 ctivated tvtd2357.42 2.355 ctivated ). State in adapted cells is particularly particularly is cells adapted α in ), the activation of ATF4, SMAD2, of ATF4, SMAD2, activation the ), α . 26,38 Thus, we examined potential potential we examined Thus, Regulation z-score -1.825 -1.654 - -1.797 -1.952 -1.921 .0 1.82 1.805 1.984 .5 1.40 1.658 1.942 1.831 1.711 1.875 . In addition to addition . In V olume 11 Issue19 P value of 8.59 4.20 3.28 8.96 1.26 5.96 1.69 2.94 1.61 7.41 overlap E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E ------07 -04 -06 -04 -04 -05 -02 -06 -08 -13 -04 -13 -15 -03 -02 - -07 -10 -16 -09 -09 -07 -08 -04 - - - 04 02 04 04 03 17 03 09 02 03 02 02 in α in -

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com depletion. of eIF2 phosphorylation reported 4A eIF2 triggered depletion Gln that revealed pathways stress ER to the data profiling RNA of the Overlapping tors. fac stress-related of ER regulation functional on depletion the of Gln effects the studied first We conditions. nutrient-insufficient different in pathways signaling ER-stress been well-characterized; have responses adaptive subsequent and ing, sens oxygen mediate mechanisms Molecular 6 Table in summarized are tions nutrient condi three all under pathways ing signal stress of ER states The depletion. Gln of that from different dramatically a pattern ( were activated pathways ATF6 ATF4, and XBP1 all conditions, tion Glc deple under hand, other On the levels. functional protein and atmRNA, repressed to be predicted was XBP1 affected, was tion or ATF6 func IRE1 that to indicate evidence media, cell growth was inhibited. was growth cell media, or Glc-free Gln-free in particularly and, tors were reprogrammed, regula cycle cell (3) and were affected; stresses ER controlling regulators transcription (2) repressed; were generally functions cell normal controlling regulators (1) transcription conditions: nutrient different in of expression gene characteristics shared several we found studies, on our Based hierarchies. and works net regulatory complicated the and pattern sion reprogramming expres gene systemic the IPAstudy andto program profiling mRNA the exploitedWe hypothesis. our test to step first our as defined, clearly be can nutrient conditions all which in system, culture cell of the advantage study, we take this In proliferation. and survival cell the to support of genes expression the regulate to and sources nitrogen and of carbon low availability the sense to ability innate an have also cells regulation, hormonal and systemic the Besides sensing. of oxygen that to similar a paradigm via availability source nitrogen and source carbon sense cells that We source. hypothesized nitrogen and source of carbon of availability change dynamic the to respond and sense how cells nent to address mental setting ( setting mental experi our in repressed to be predicted was upregulated genes under each nutrient condition, but also by the by the nutrient but condition, also each under genes upregulated the notby only reflected was difference This features. tinctive dis has condition each under occurred that expression gene the inhibition, growth Glccause depletion and depletion Gln both ent condition (log condition ent nutri this under decreased dramatically was level ATF4 mRNA that fact to the ascribed ), which is consistent with our previously previously our with consistent is which ), 14 However, functionally, ATF4 However, functionally, Discussion Fig. 4A Fig. 2 Ratio = -6.13). While no While =-6.13). Ratio ); this result might be be might result this );

18 activation ( α activation Fig. 4B Fig. it becomes emi it becomes . upon Gln α upon Gln 14 ), showing showing ), More importantly, although although importantly, More Fig. Fig. ------control, and mRNA levels in the control sample were arbitrarily defined as 1. defined arbitrarily were sample control the in levels mRNA and control, by qR by determined were mRNA of Levels validation. further for up picked were analysis microarray by upregulated be to identified genes Representative MM01 in cells. genes selected of tion upregula- the of Validation (C) 0.084). (p = insignificant statistically remain genes of group this levels of some regulators of G of regulators some of levels expression the that Note affected. significantly not were genes of groups same the hand, other the given).On were values p (-log reprogrammed significantly were genes regulatory cycle cell of groups four all cells, Gln-depleted acute In MM01 cells. and Hep3B Gln-depleted acute in regulators cycle cell of analysis (B) Functional cells. Gln-depleted acute in downregulated 43 while upregulated, found were > them, 166 Ratio 1.0).Among (log2 upregulated as fied identi- were 292 MM01genes In cells, cells. Gln-depleted acute in states expression their and MM01 in genes (A) Upregulated media. toGln-free have adapted MM01 cells cells, depleted Figure 3. T - P C Gene expression and cell cycle regulation in MM01 cells. In contrast to acute Gln- toacute contrast In MM01 in cells. regulation cycle cell and expression Gene R. RNA samples isolated from Hep3B cells cultured in complete media were used as as used were media complete in cultured cells Hep3B from isolated samples RNA R. C ell Cycle - - - - uptake of glutamate (Glu) by most types of cells, Gln depletion depletion Gln of cells, types by most (Glu) of glutamate uptake 5 Figure in shown of roles Gln metabolic the Considering by cells. synthesized be can acids amino essential non- that concept of the because partly past, the in studied sively inten not been has acids amino However, of non-essential lack starvation. acid amino essential from obtained on results based progression. cycle cell or Glc restricts Gln were indispensible nutrients for cell cycle progression through G through progression cycle for nutrients cell were indispensible Gln Glc and both that demonstrated for 18 data their h, and media or Gln-free Glc-free in cells HeLa synchronized cultured phase. nized HeLa cells in G in cells HeLa nized of non-synchro arrest the Glccauses depletion and Gln both we found and manner, Gln-dependent in to grow demonstrated been has which line cell another HeLa, we used trypsinization, during clumps form easily Hep3B cells Because progression. cycle or of Glcon depletion Gln cell effects the tested we also Moreover, factors. of transcription response functional predicted

Amino acid starvation response has been studied, primarily primarily studied, been has response starvation acid Amino 39 1 /S checkpoint in MM01 were affected, but collectively, change of change collectively, but affected, MM01 in were /S checkpoint Therefore, we conclude that the low availability of either low availability the we conclude that Therefore, 2 /M phase. In a recent paper, Sergio et al. al. et paper, arecent In Sergio phase. /M and the inefficient inefficient the and 3685 - - 1

©2012 Landes Bioscience. Do not distribute. 3686 line to insufficiency. long-term Gln line cell adapted an as ammonia supplemented with media Gln-free in surviving MM01 cells utilized also we insufficiency, Gln acute to addition In biosynthesis. its activate and acid amino essential non- of a specific lack to the responding and sensing cells’ tumor in participate also pathways other if However,tion. it unclear is eIF2 caused also depletion 24 hGln Indeed, cysteine. intracellular of to lack leading of cystine, uptake the reduces turn, Glu, in decreased Glu, and and of Asn levels decreased in result also will may trigger general inhibition of translation via eIF2 via of translation inhibition general trigger may α (ER ESR1 and SMAD3 Moreover, response. SMAD2, of ER-stress establishment successful suggesting MM01 in cells, activated study, and were DDIT3 this found we ATF4 In insufficiency. to Gln response cellular involved in regulators critical identify may conditions both in upregulated genes on the studies ther fur MM01 cells; adapted in and Hep3Bcells Gln-insufficient acute in upregulated genes the paper, compared we only this In not shown). out die (data eventually and phenotype liferative show more pro slowly will MM01 removed, is cells ammonia if complete media; regular in cultured Hep3B with cells compared Functional responses of transcription regulators in MM01 in cells regulators transcription of responses Table 5. Functional (A) Functional states of of states (A) Functional analyzed. set data the in covered not factors non-filled and inhibited filled green activated; filled red change; significant no indicate factors Grey-filled P I by generated were of States page). opposite 4(See Figure Transcription E Regulator R

T MYOD1 α eIF2 of tophosphorylation K leads MD ciae 2.845 Activated SMAD2 MD ciae 2.161 Activated SMAD3 MD niie -2.239 Inhibited SMAD7 P phosphorylation ( α phosphorylation NCOR1 N1 niie -2.814 Inhibited HNF1A N4 niie -3.135 Inhibited HNF4A PP FB niie -2.025 Inhibited NFKB1 P GA C DDI FA TP E A I1Atvtd2.124 Activated HIC1 R ) were predicted to be functionally activated, suggesting suggesting activated, functionally to be ) were predicted P L FOS E R ciae 2.382 Activated SR1 T DM1 AR AG1 GR B T P 3Atvtd2.152 Activated 73 4Atvtd2.755 Activated F4 4I A4 T P AIhbtd-2.024 Inhibited 2A I A ciae 2.302 Activated A ciae 2.268 Activated 3 Predicted P hbtd-2.081 nhibited hbtd-2.170 nhibited A State . E T R h -stress signaling pathways in Gln-depleted cells. (B) Functional states of of states (B) Functional cells. Gln-depleted in pathways signaling -stress ree transcription factors, A factors, transcription ree Fig. S2 Fig. Regulation ), z-score , hence translationally activating A activating translationally , hence -1.863 14 -1.961 -1.752 1.664 1.757 1.913 E 14 indicating Gln depletion depletion Gln indicating R MM01 cells grow slowly slowly grow MM01 cells -stress signaling pathways under Gln- and Glc-depleted cells. Changes of of Changes cells. Glc-depleted and Gln- under pathways signaling -stress T F 4, A 4, p value of T overlap 6.86 4.60 3.33 2.99 4.97 4.30 9.95 3.33 6.03 1.97 1.45 2.69 1.77 2.19 2.43 1.03 1.91 4.43 5.54 2.71 1.97 F6 a α activa E E E E E E E E E E E E E E E E E E E E E nd XB nd - - - -07 -11 -03 -13 -04 -02 -02 -02 - -02 - -03 - -03 -07 -02 -18 -17 02 02 03 02 06 04 C P ell Cycle 1 , have been reported to play critical roles in in roles critical toplay reported , have been - - - T F 4. 4. unfolded protein response, unfolded by ATF6activated is that reported been It depletion. by has Gln triggered that from different stress acellular triggered depletion Glc that depletion, suggesting by Gln caused that from different ( of pathway XBP1 activation functional and upregulation for the responsible partly be may of which ATF6, activation functional astrong observed study, our In we of Glc (or G6P). lack to the respond directly cells how other unknown However, been it release. has and duction pro insulin the mediates which ATP levels, the affects directly phenotype. slow-growing to the contributing ability, thus avail Gln decreased the to match rate proliferation cell the uate Gln-free DMEM with 10% dialyzed FBS and 4 mM Gln for Gln 4mM and FBS dialyzed 10% with DMEM Gln-free in cultured and of 1:3 aratio in study, were passed Hep3B cells For glutamine-deprivation acute isolation. for RNA 24 h before medium fresh in were cultured day, next cells the of 1:3, a ratio in passed were split study, and MM01 cells tamine-deprivation Forglu long-term confluence. 90% reaching when were passed cells and 2d, every changed was medium culture the sphere and MM01 cells were kept at 37°C in a humidified 5% CO 5% ahumidified in at37°C were kept MM01 cells ammonia. mM 0.8 and 10 kD) dialyzed Biologicals, (Atlantic FBS dialyzed 10% with (15-0130-CV) DMEM Gln-free in cells Hep3B by culturing were established MM01independent cells and HIC, Hep3B adaptation to Gln insufficiency. Hep3B to Gln adaptation involvementthe of TGF CO 5% ahumidified in at37°C (FBS) serum bovine fetal 10% with supplemented medium essential minimal Eagle’s in were cultured (ATCC Hep3B HB-8064) cells reagents. and media culture Cell treatment. for tumor targets provide putative may molecules of these knocking-down or Inhibiting nutrient-deprived in conditions. survival cell tumor facilitate that molecules the It to identify possible is conditions. nutrient to poor more adaptive cells cancer by selecting processes oncogenic in participate also may that responses adaptive and stresses cellular in results Gln of Glc and availability Imbalanced hierarchies. regulatory but not identical, related, through death cell eventual and progression cycle of cell inhibition to the lead of will Glc either or Gln Lack roles. metabolic distinctive has but each proliferation, and survival for nutrients cell important Glc two represent and Gln that revealed conditions those under patterns expression gene of the Analysis nutrient conditions. underdefined of Hep3Bcells database profiling expression protein stress. unfolded from to prevent cells E

R s cells, the supply of the Glc βcells, pancreatic well-studied the In In conclusion, we have established a genome-wide gene gene agenome-wide conclusion, established we have In 2 tress also activates IR activates also tress atmosphere, and the cells were passed twice a week. Gln- aweek. twice were passed cells the and atmosphere, 41,42 which encode two tumor suppressors, might atten- might suppressors, tumor two encode which E Materials andMethods R Fig. 4B Fig. -stress signaling pathways in Glc-depleted cells. Glc-depleted in pathways signaling -stress E 1 , which facilitates the splicing of XB of splicing the facilitates , which and estrogen signaling pathways for pathways signaling estrogen β and 43 ). This ER-stress response pattern is is pattern response ER-stress This ). suggesting Glc supply is important Glc supply important is suggesting E E R R -stress response. Activation of of Activation response. -stress -stress signaling pathways pathways signaling -stress 40 The activation of TP73 of TP73 activation The V olume 11 Issue19 P 1 R 2 atmo NA. NA. - - - -

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com Figure 4. For figure legend,see page 3686.

C ell Cycle

3687

©2012 Landes Bioscience. Do not distribute. 3688 was extracted from the cells using RNeasy Mini Kit (Qiagen). (Qiagen). Kit Mini RNeasy using cells the from extracted was Total RNA PBS twice. ice-cold with washed then for 24 hand controlcells. as used were Gln 4mM and FBS dialyzed 10% with DMEM Gln-free in cultured Hep3B isolation. cells RNA for 24 hbefore FBS) dialyzed 10% with DMEM (glucose-free medium fresh in tured were cul day, next cells the for adaptation; FBS dialyzed 10% with DMEM glucose-free in cultured and of 1:3 aratio in passed study, were Hep3B For glucose-deprivation cells acute isolation. RNA for 24 hbefore FBS) dialyzed 10% with DMEM (Gln-free medium fresh in were cultured day, next cells the adaptation; **Adapted to Gln-free media, long-term survival and proliferates at slow rate. slow at proliferates and survival long-term media, toGln-free **Adapted States of of States Table 6. *Based on eIF2 on *Based cells with A with cells Figure 5. Glc utilization work together to facilitate the biosynthesis of nitrogenous biomolecules. nitrogenous of biosynthesis the tofacilitate together work utilization Glc and Gln source. acarbon as cells by utilized eventually be may Glu or Gln of skeleton carbon Finally, the cystine. touptake cells for energy the provides gradient concentration the down Glu of release the example, for nutrients; other touptake cells for important isalso Glu of tion concentra intracellular high a relatively Maintaining molecules. nitrogenous other and amine poly- glutathione, nucleotides, proteins, of synthesis the for substrate as serves also Glu tion, addi- In acids. amino non-essential other of biosynthesis the in group amino of donor major toα transferred be can acids amino other from groups amino redistribution, and storage nitrogen of hub acentral as serves Glu ciently. effi- cells by taken be cannot which Glu, of aprecursor as serves also It nucleotides. and Asn of synthesis the for isasubstrate Gln activities. anabolic for skeletons carbon and power ing RNA isolation.RNA

Glc (-),24h Gln(-), 24h Nutrients MM01** Distinct metabolic roles of Glc and Gln in cell proliferation. Glc utilization provides provides utilization Glc proliferation. cell in Gln and Glc of roles metabolic Distinct T P , N phosphorylation detected in western blots, all other predication was based on I on based was predication other all blots, western in detected α phosphorylation AD E R P MM01 and Hep3B cells were treated as above above as were treated MM01 Hep3B and cells -stress signaling pathways at various nutrient conditions nutrient various at pathways signaling -stress H a nd carbon metabolites, which fulfill the major needs of energy, reduc- energy, of needs major the fulfill which metabolites, carbon nd +NH 4 + -KG to form Glu. On the other hand, Glu serves as a as serves Glu hand, other the On Glu. toform -KG Activation ciainAtvto ciainAtvto Arrest/death Activation Insignificant Activation Insignificant Activation Activation Activation Activation *eIF2α C niiinIsgiiatIhbto Arrest/death Inhibition Insignificant Inhibition ell Cycle ATF4 - cut-off during upload, if not otherwise specified. (3) Biostatistic Biostatistic (3) specified. not otherwise if upload, during cut-off differentially expressed genes, log genes, expressed differentially for analysis; comparative 0.05 for further were included than less p value detected with genes group; for each data microarray (1) Filter the processes: basic the are biology. following The lian mamma on findings published on previously based networks molecular and pathways to canonical ontology,gene relevance to according network or metabolism function gene to generate used be IPA Inc.). can Systems, Ingenuity www.ingenuity.com (IPA, analysis pathway Ingenuity using analyzed was set data ray each observation, log observation, each IPA to data Upload (2) for data. significant as were included

- ATF6 P A a to as referred numbers, cycle (threshold data collected from were made studies (RQ) tion quantifica Relative loading. of RNA ization for gene normal ahousekeeping as used was Biosystem). TaqMan (Applied probes gene-specific validated using analysis titative for quan of 1/50used ratio and with diluted was cDNA Then cDNA quality. confirm for β PCR (Invitrogen). (Applied Biosystems). (Applied using SuperScript II Reverse Transcriptase Transcriptase Reverse SuperScript II using cDNA to reverse-transcribed first was RNA Total genes. selected of confirmation PCR) or < 0.05 and displayed as log as displayed 0.05 and < value as ap were defined sion differences expres Significant Corp). (Omicsoft Studio Array using analyzed was data GenePix The Pro version 4.0 (Axon). by GenePix processed and measured was intensity signal and chips, array with were hybridized MM01and cells Hep3B from samples The DNA elements. sense-strand 60-mer as formed probes trol con 1,088 and experimental genome probes 29,187 32,275 oligonucleotides, tain human con OneArrays Human Group). Biotech (Phalanx by OneArrays performed was filing nm. Technologies) at260/280 (Nanodrop spectrofotometer Nanodrop using determined was concentration RNA nalysis. 2 Ratio values > 1.00 or <-1.00 values as were used Ratio The microar The analysis. pathway Ingenuity Quantitative real-time PCR (qRT- PCR real-time Quantitative Gene expression pro expression Gene analysis. Microarray

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C t) using StepOne™ Software v2.1 StepOne™ t)Software using XBP1 2 Ratio values with p<0.05 with values Ratio 2 -actin was used to used was -actin ratio values >1.00 values ratio V olume 11 Issue19 Cell Fates Adapted β -actin -actin ------

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com NaCl, 0.2% Tween 20) and probed with anti-Phospho-eIF2 Tween with probed NaCl, 0.2% and 20) Tris, (50 mM pH 8.0, 150 buffer TBST powder in milk dry nonfat 5% with blocked (Bio-rad), membrane PVDF Immunoblot onto were transferred proteins separated protocol. The facturer’s manu the following run and (Bio-rad) gel precast protein TGX into a Mini- loaded of protein was amount Same mix. inhibitor DTT,SDS, 5mM 1% Triton X-100, 1x protein and Glycerol 10% 1% 10 TRIS-HCl, mM Murea, 6.6 containing buffer lysis urea for presentation. IPA from reformatted and exported and generated were then networks and data The systems. Ingenuity within integrated algorithms one several of the algorithm, sis analy core the using built were then experiments our from 11904312, 2011-12-15). Date: Release networks Hypothetical version: (Content IPA into uploaded the program was set data Our association. of casual alower probability means score higher the pathways; canonical the with data of our association casual the reflect scores These score). (Z scores network Calculate (4) significance). 0.01 biostatistical as than less pvalues (it sets factor by atranscription regulated genes the and set data uploaded an in genes the between overlap significant measures pvalue laping over test; Fischer’s by the pathway exact canonical the and data our between association of the pvalue) (overlap probability the determining (b) or pathway canonical one in specific ofber genes num total of the regard in data microarray our from of genes age percent the calculating (a) through pathways canonical known the and data microarray our between association of the analysis 7. 6. 5. 4. 3. 2. ang CV. Glutaminolysis: supplyingcarbonornitro- 1. 8.

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mM mM C ell Cycle α - - - - - 0.05 for each analysis. 0.05 for each at set was level significance The data. to analyze used t-test was Student’s deviation. ± standard mean Values as tions. were shown analysis. of the validity statistical to maximize were collected of events 10,000 Aminimum laser. 488-nm a with Biosciences) Dickinson (Becton cytometer flow a with FACScan cytometry by flow analyzed was sample Each propidium iodide. using stained and were trypsinized cells Then, or Glc for Gln either 18 without or medium h. complete medium scientific). (Thermo system chemiluminescence with bined com antibody secondary (HRP)-conjugated peroxidase radish horse using were visualized bands antibody, and (Sigma-Aldrich) funds from Drexel University. Drexel from funds start-up and (NIH) of Health Institutes NCI, National from R01-CA129494 by grant (to NS) part in work supported is This disclosed. were interest of conflicts No potential eIF2 total Signaling), (Cell www.landesbioscience.com/journals/cc/article/21944/ here: found be may materials Supplemental

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