Growth Factor Receptor Binding Protein 14 Inhibition Triggers Insulin

Growth factor receptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is associated with hepatocellular carcinoma Lucille Morzyglod, Michèle Caüzac, Lucie Popineau, Pierre-Damien Denechaud, Lluis Fajas, Bruno Ragazzon, Véronique Fauveau, Julien Planchais, Mireille Vasseur-Cognet, Laetitia Fartoux, et al.

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Lucille Morzyglod, Michèle Caüzac, Lucie Popineau, Pierre-Damien Denechaud, Lluis Fajas, et al.. Growth factor receptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is associated with hepatocellular carcinoma. Hepatology, Wiley-Blackwell, 2017, 65 (4), pp.1352-1368. 10.1002/hep.28972/suppinfo. hal-01606970

HAL Id: hal-01606970 https://hal.archives-ouvertes.fr/hal-01606970 Submitted on 25 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is doi: 10.1002/ hep.28972 differences betweenthisversionand theVersionofRecord. Pleasecitethisarticle as through thecopyediting, typesetting,paginationandproofreading process whichmayleadto This articlehasbeenaccepted forpublicationandundergonefullpeer reviewbuthasnotbeen et des Sciencesl’Environnement dede Paris, Bondy, 93140 France F-75013 Paris,F-75013France F-75012Paris,(CRSA), France 11 10 9 8 7 6 5 4 3 2 1

France Lucille Morzyglod Lucille Guilmeau Vasseur-Cognet Anne-Françoise Burnol Fajas Luis Grb14 de laInstitut Santé Nationalla deRecherche et Médicale,FranceParis, Université Paris Descartes,Cité, SorbonneParis France CNRS, UMR8104,Paris, France U1016,Inserm, Institut Paris,Cochin, France Center Integrative for Genomics,Lausanne, Universityof LausanneCH-1015,Switzerland Department Physiology, of Lausanne,University of LausanneCH-1015, Switzerland APHP,HôpitalLa Pitié Salpêtrière,d’Hépato-Gastroentérologie, Service Paris, F-75013 Sorbonne Universités, France Paris, UMR242,IRD UPEC, UPMCCNRS7618, 113,INRAParis71392,113, d’Ecologie Institut APHP, Hôpital LaAPHP, HôpitalService Pitié-Salpêtrière, de Chirurgie Hépatobiliaire Transplantation, et Universités,Sorbonne UPMC Univ 06, INSERM, Paris Centrede RechercheSaint-Antoine

inhibition triggers insulin-induced mousehepatocy insulin-induced triggers inhibition 1,2,3 4,5 Cteie Postic Catherine , Buo Ragazzon Bruno , 6,7,8 1,2,3 Leii Fartoux Laetitia , Comment citer cedocument: , Michèle Caüzac Michèle , This article isprotected by copyright. All rights reserved. associated with hepatocellular carcinoma carcinoma hepatocellular associated with

1,2,3 *

1,2,3 1,2,3 Catl Desdouets Chantal , Vrnqe Fauveau Véronique , 9,10 1,2,3 , Olivier Scatton Olivier , , Lucie Popineau Lucie ,

10,11 1,2,3 1,2,3 1,2,3 Oiir Rosmorduc Olivier , Crsèe Desbois-Mouthon Christèle , Jle Planchais Julien , , Pierre-Damien Denechaud Pierre-Damien , te proliferation and is proliferation te 1,2,3 9,10 Mireille , Sandra , 4,5 7 , , Version postprint Page 3of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is pourFondation la Recherche Médicale. a by d (Comité Cancer le contre Ligue the and DGOS_5790) In the from grants by supported was Center Research work The Equipe). (Labélisation Médicale Recherche and ANR-09-JCJC-0057) ANR-10-Wnt-Metaboliv, Grb14, n Agence the from grants by supported was work This Financial supports r of target proliferating nuclear cellantigen ;PI3K:phospha mechanistic mTORC1 : ; receptor insulin inducible iLIRKO : carcinoma ; hepatocellular HCC : ; 14 protein binding receptor factor Growth :Grb14 knockout S6K double DKO : bromodeoxyuridin; :BrdU Listabbreviations of haveThe conflictauthors nointerest. of +33153732709,tel. : +33144412421, fax : E-mail : Anne-FrancoiseDr. Burnol Contact information: S6K;Keywordsinsulin : E2F1 ; signaling IR-A/IR- ;

Hepatology Hepatology tidylinositol-3Rb ; :retinoblastoma. kinase B ;adenoviral B shRNA [email protected] SA:gn e nihet analysis ; enrichment set gene GSEA : ie isln eetr ncot; IR : knockout ; receptor insulin liver e Paris). LM and LP were supported were LP and LM Paris). e efre wti te Saint-Antoine the within performed toae e a ehrh (ANR-06- recherche la de ationale ; EGF : epidermal growth factor factor ; growth epidermal EGF : ; ttt ainl u acr (INCa- Cancer du National stitut pmcn ope 1; PCNA : 1; complex apamycin P a a elwhp rm the from fellowship a had LP y h Fnain or la pour Fondation the by 2

Page 4of54 Version postprint Page 5of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

lrfe (2 1) Isln inln ad cin ar action and signaling Insulin 13). (12, clarified and cycle t cell regulates and characterized, mitogenesis stimulates well are actions metabolic induced mo the While 11). (10, degrees variable to involved Ras-ERK1/2 and PI3K-Akt Ras-E type, cell the the on depending by triggered be to considered classically ca signaling PI3K-Akt the by mediated predominantly pathways. Ras-ERK1/2 the and (PI3K)-Akt PI3-kinase progressiongrowthand (6). li in implicated directly de be can IR that the suggesting to IR-A of overexpression an showed recently mitog more be to considered is and cells tumor many I and insulin, of effects metabolic the in involved isoforms splicing two as exists IR 6). (5, process I that suggesting tumors, human of types several in expressio the Furthermore, 4). (3, candidate likely develo which hyperinsulinemia, cancer, and diabetes t factors the Among (HCC). carcinoma hepatocellular developing of risk higher at are diabetes 2 type or studies Epidemiological (2). cancer of development documented poorly direct a However, types. cell many in behaves proliferation insulin actions, metabolic its to addition glucose maintain to tissues target metabolic its on circ increased to response in cells beta pancreatic nui siuain fteI atvts w an in main two activates IR the of stimulation Insulin h metabolic controlling hormone unique a is Insulin Comment citer cedocument: n vivo in This article isprotected by copyright. All rights reserved. atog is novmn i icesnl suspecte increasingly is involvement its although , Hepatology Hepatology R-A which is expressed in fetal cells and in and cells fetal in expressed is which R-A : IR-B which is mainly expressed in tissues in expressed mainly is which IR-B : ver neoplastic transformation and/or tumor and/or transformation neoplastic ver and dying from multiple cancers, including cancers, multiple from dying and n of the insulin receptor (IR) is deregulatedis (IR) receptorinsulin the of n ulating blood glucose levels, insulin acts acts insulin levels, glucose blood ulating R signaling is involved in the tumorigenic the in involved is signaling R n lpd eaoi hmotss 1. In (1). homeostasis metabolic lipid and fet f nui o cl poieain is proliferation cell on insulin of effect highlighted that individuals with obesity obesity with individuals that highlighted e also tightly monitored by negative negative by monitored tightly also e ps secondary to insulin resistance, is a is resistance, insulin to secondary ps 1S rgeso rmi t b fully be to remain progression G1/S s got fco siuaig cell stimulating factor growth a as nc hn RB 7. neetnl, we Interestingly, (7). IR-B than enic eua ptwy mdaig insulin- mediating pathways lecular inln ptwy ae iey o be to likely are pathways signaling a ae ucpil t ln obesity, link to susceptible are hat cd, n te ioei efc is effect mitogenic the and scade, K/ ptwy 8 9. However, 9). (8, pathway RK1/2 e ehnss hrb insulin whereby mechanisms he rclua inln ptwy, the pathways, signaling tracellular rmn o I- i hmn HCC, human in IR-B of triment Metabolic actions of insulin are are insulin of actions Metabolic motss Scee b the by Secreted omeostasis. i the in d 4

In this study we demonstrate that Grb14 expressionstudyGrb14wedemonstratethis that In Comment citer cedocument: This article isprotected by copyright. All rights reserved. GRB14 Grb14 xrsin s infcnl dcesd n ua HCC human in decreased significantly is expression deletion also enhances insulin-induced cell growth cell insulin-induced enhances also deletion Hepatology Hepatology n inhibitor of IR catalytic activity, and that its that and activity,catalytic of IR inhibitorn for insulin signaling in the development of development the in signaling insulin for itogenic and metabolic effects of insulin of effects metabolic and itogenic 9, 14). We previously reported that that reported previously We 14). 9, nln ad h R/21 pathway. Rb/E2F1 the and gnaling rvl ht ontem h IR-B the downstream that nravel is required to protect hepatocytesprotect required to is cs pouto i cultured in production ucose in 5 , ,

Page 6of54 Version postprint Page 7of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is ih h Fec lw ad euain (NL ubr 1 number (CNIL regulations and laws French the with S department, (Pathology biobank tumour a in stored f resection liver curative undergoing patients from Patients andPatients liver tissue specimens infrozen liquidnitrogen at stored°C −80 and unti analysis immunohistochemistry for formalin buffered part collected, was Blood (Sigma). BrdU of mg/kg 50 sacrif to prior hours two and injection, adenoviral Invivogen). mg/kg, (4.5 Rapamycin or Aldrich) Sigma eithe sacrifice before hours) 2 and hours (18 twice inhibitor with experiments For serum. physiological S6K1 of Generation deletion. IR after weeks three Fift (20). previously described were mice knockout) stud before week one Harlan for from purchased were environment mice male Nine-week-old the to adapted Animalstreatments and EXPERIMENTAL PROCEDURES Ui o a hN targeting shRNA a or (USi) the penis vein with 2.10 with vein penis the with anesthetized were Mice Paris”. de vétérinaires “Dire the by approved were and animals experimental French the to according out carried were procedures carbohyd (65% diet regular and water to access free temp a in cycle light/dark 12-h a with cages colony labo Jackson from were mice KO E2F1 (21). described Eighty-five HCC (T) and paired adjacent non-tumour adjacent paired and (T) HCC Eighty-five Comment citer cedocument: This article isprotected by copyright. All rights reserved.

9 pfu of recombinant adenovirus expressing either a either expressing adenovirus recombinant of pfu Grb14

Gb4) 1) n fnl oue f 150 of volume final a in (18) (Grb14i) Hepatology Hepatology l used. l used. ice they were intraperitoneally injected with injected intraperitoneally were they ice or HCC at the Saint-Antoine Hospital and Hospital Saint-Antoine the at HCC or r with Akt inhibitor VIII (Akti, 50 mg/kg, mg/kg, 50 (Akti, VIII inhibitor Akt with r erature-controlled environment and had and environment erature-controlled sfuae eoe h ijcin through injection the before isoflurane , ie ee netd intraperitoneally injected were mice s, . The remaining liver tissue was flash- was tissue liver remaining The . e-ekod ae ie ee studied were mice male een-week-old of liver tissue was fixed in 4% neutral neutral 4% in fixed was tissue liver of Mice were sacrificed two days after after days two sacrificed were Mice -/- udlns o te ae n ue of use and care the for guidelines itAtie optl i accordance in hospital) aint-Antoine ae 1% a ad 4 poen. All protein). 24% and fat 11% rate, /S6K2 to dpreetl ds services des départementale ction y. The iLIRKO (inducible liver IR IR liver (inducible iLIRKO The y. aois Al ie ee osd in housed were mice All ratories. 913901 v0). Part of this tissue tissue this of Part v0). 913901 (NT) liver tissues were collected were tissues liver (NT) -/- Laboratories (C57BL6/J) and (C57BL6/J) Laboratories ie a be previously been has mice control shRNA control sterile l 6

ie, rti ad mRNA and protein lines, 7

Page 8of54 Version postprint Page 9of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is BrdU labelling was similar in USi and Grb14i mice l mice Grb14i and USi in similar was labelling BrdU infec adenoviral after days two peaking transiently division Hepatocyte (Fig.1F). increased moderately after level protein and H3 (phospho-histone mitosis or ( te cl tps ee aeld Acrigy severa Accordingly, labelled. were types cell other animals. USi control the hepatocytesofpha 33%Sinjection, werein the sacrifice. the before hours two BrdU with injected content. prom the thus is proliferation cellular in involved cyc KEGGcell the with especiallypathways (Fig.1C) by altered genes between s (GSEA) analysis enrichment set Gene and performed (GSK3 downst the of and Akt and ERK1/2 of phosphorylation sig insulin improved significantly respe This levels protein(Fig.1A-B). and mRNA the at 30% and 40% by 48 after wereanalyzedlivers and (USi) RNA control eit with injected were Mice strategy. adenoviral an downregulated we proliferation, hepatocyte Liver specific down-regulationofexpression Grb14 RESULTS ccnb1 β, )) which control the progression of the cell cycle, cell the of progression the control which )) To validate the transcriptomic data on the prolifer the on data transcriptomic the validate To o xeietly netgt te osqecs f a of consequences the investigate experimentally To S6K, 4EBP1) (Fig.1B, Supporting Figure S1A). A liv A S1A). Figure Supporting (Fig.1B, 4EBP1) S6K, Grb14 Comment citer cedocument: In situ In This article isprotected by copyright. All rights reserved. oneuain Fg1) B cnrs, yln 1 ( D1 cyclin contrast, By (Fig.1F). downregulation BrdU staining was mainly detected in hepatocytes, in detected mainly was staining BrdU Grb14 oneuain n bt cl cce n DA replica DNA and cycle cell both and downregulation Ki67 Hepatology Hepatology wr srknl icesd t RA ee and/or level mRNA at increased strikingly were ) Grb14 inent feature after a decrease in Grb14 liver Grb14 in decrease a after feature inent expression in liver of C57Bl/6 mice using mice C57Bl/6 of liver in expression tion (Supporting Fig.S1B). From day four, four, day From Fig.S1B). (Supporting tion As shown in Fig.1E, 48h after the Grb14i the after 48h Fig.1E, in shown As induces hepatocyteproliferation her Grb14 adenoviral shRNA (Grb14i) or (Grb14i) shRNA adenoviral Grb14 her se of the cell cycle, as compared to 2% into cellcomparedcycle, theas of se h. Grb14 liver expression was decreased expressionwasliver Grb14 h. le gene set (Fig.1D). Activation of genes of Activation (Fig.1D). set gene le iver, while concomitantly Grb14 protein Grb14 concomitantly while iver, ccis E ( (E cyclins l nue by induced aig a son y h enhanced the by shown as naling, and markers of proliferation (PCNA) proliferation of markers and howed a robust positive association positive robust a howed em fetr o te k pathway Akt the of effectors ream ation state of liver cells, mice were mice cells, liver of state ation aue elto o Gb4 on Grb14 of depletion acute n ctively in Grb14i-treated mice mice Grb14i-treated in ctively er transcriptomic analysis was analysis transcriptomic er Grb14 ccne , ( A ), oneuain was downregulation ccnd1 ccna2 and very few few very and ) was only only was ) ad B1 and ) tion 8

in 9

Page 10of54 Version postprint Page 11of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is of mTORC1, we then used S6K DKO mice, which are del are which DKOS6K mice, used wethen mTORC1, of EP sgaig ahas ee rsre (i.C. S (Fig.5C). preserved were pathways signaling 4EBP1 totallywas expression S6Kexpected as While (21). ( E cyclin while suggesting theninsulinathat act as powerful can IR i incorporation BrdU in increase The (Fig.3B). (23) stimu reported previouslytoaccording the control, Basal(20) Grb14 (Fig.3A). levels protein and mRNA eaoye iiin nue b Gb4 iecn i m is silencing Grb14 by induced division Hepatocyte Grb14 cyclin A ( A cyclin labelling BrdU withcorrelation In shown). not data the by induced staining hepatocyte BrdU in increase mTORC1 of or (Akti) AktiVIII by Akt of wortmannin, pharmacologica a used first we division, hepatocyte signaling pathway o IR to ( of effect aft increased and mice iLIRKO in detected still was activa not were and knockout IR by blunted severely evid provides This Grb14i. with or USI with treated striking was S6K and Akt ERK1/2, of Phosphorylation ccne1) lox/lox lox/lox oneuain a a lat aty trbtbe to attributable partly least at was downregulation mice injected with Grb14i was markedly decreased b decreased markedly was Grb14i with injected mice o vlae h cnrbto o te PI3K-Akt-mTORC1 the of contribution the evaluate To , cyclin A ( A cyclin , ie Fg3-) Tee aa niae ht hepatocy that indicate data These (Fig.3D-E). mice ccna2 Grb14 ccne1) ), cyclin B1 ( B1 cyclin ), oneuain on downregulation ccna2 Comment citer cedocument: This article isprotected by copyright. All rights reserved. was not significantly altered (Fig.4B-C). To furth To (Fig.4B-C). altered significantly not was ) and cyclin B1 ( B1 cyclin and ) ccnb1 ) and ) Ki67 Hepatology Hepatology Ki67 ccnb1 xrsin n o te xrsin f yln E cyclin of expression the on and expression was severely inhibited by Akti and rapamycin, and Akti by inhibited severely was ) was inhibited in liver from iLIRKO compared compared iLIRKO from liver in inhibited was ) mitogen in liver.mitogen the latory effect of insulin on insulin of latoryeffect , Grb14i-mediated increased expression of expression increased Grb14i-mediated , ence that insulin signaling pathways were pathways signaling insulin that ence n hepatocytes nuclei observed in control in observed nuclei hepatocytes n inhibited in S6K DKO mice liver, Akt and Akt liver, mice DKO S6K in inhibited werelowercompared in iLIRKO livers to ted by Grb14i treatment, while p-4EBP1 p-4EBP1 while treatment, Grb14i by ted er by rapamycin significantly reduced the reduced significantly rapamycin by downregulation of of downregulation ly reduced in liver from iLIRKO mice mice iLIRKO from liver in reduced ly Grb14 apoc. niiin f IK by PI3K of Inhibition approach. l dae b te PI3K-Akt-mTORC1 the by ediated eted for both both eted for K ee eein eue the reduced deletion gene 6K h atvto o I signaling, IR of activation the y the deletion of the IR (Fig.3C). IR the of deletion the y downregulation (Fig.3D). The (Fig.3D). downregulation aha i Grb14i-induced in pathway e rlfrto idcd by induced proliferation te S6k1 er investigate the role role the investigate er Grb14 Grb14 and (Fig.4A and (Fig.4A S6k2 expression genes 10 Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is Akt-mTORC1 the that suggesting treatment, rapamycin of downregulation the by induced expression protein p Rb the in increase of the activation Interestingly, downregulation. an supporting (Fig.6D), (25) TKO) the of deletion enrich the by downregulated significant or upregulated a showed GSEA (Fig.6C). activity and activity, E2F induced treatment mitogenic hepatoc primary transfected in gene reporter E2F-RE enhancedtranscriptional(Fig.6A-B).Theactiv also prima in as well as liver mouse in treatment Grb14i serine different on Rb of phosphorylation The (24). gene cyclin induce to E2F allowing phosphorylation, b activity, their repressing factors, transcription poin restriction G1 the controls which co-repressor a protei retinoblastoma the of We status phosphorylation division. hepatocyte Grb14i-dependent mediates by caused expression Theincrease in incorporation nuclear hepatocyte BrdU of induction ucinl bEF ptwy s rca fr hepatocyt for downregulation crucial is pathway Rb/E2F1 Functional pathway. Grb14 in proliferation hepatocyte that suggest data liver, while ( E cyclin h nx se ws o dniy h ptwy downstrea pathway the identify to was step next The

Ki67 Comment citer cedocument: ccne1) expression and cyclinandA ( expression This article isprotected by copyright. All rights reserved. Grb14 expression wasnot mRNA Altog modified (Fig.5C-D). oneuain a as sgiiaty niie in inhibited significantly also was downregulation Hepatology Hepatology ccna2 Grb14 ity of E2F factors was investigated usingainvestigatedE2Fwas of factors ity t. Unphosphorylated Rb binds to the E2F the to binds Rb Unphosphorylated t. t h cmlx s irpe atr Rb after disrupted is complex the ut n (Rb), a well characterized transcription characterized well a (Rb), n ry hepatocytes, and Rb protein level was level protein Rb and hepatocytes, ry ) and cyclin and ) ( B1 expression and cell cycle progression cycle cell and expression opoyain n EF mN and mRNA E2F1 and hosphorylation ytes. Stimulation of hepatocytes by the by hepatocytes of Stimulation ytes. fe te r1i ramn (Fig.5A-B). treatment Grb14i the after eius a srknl icesd by increased strikingly was residues he mmes f h R fml (Rb family Rb the of members three downregulation further enhanced this enhanced further downregulation i liver is mediated by the Akt-S6K Akt-S6K the by mediated is liver i pathway was involved in Rb-E2F1 Rb-E2F1 in involved was pathway

et n r1i ie fr genes for liver Grb14i in ment bEF aha upon pathway Rb-E2F Grb14 dvso idcd y Grb14 by induced division e aye te xrsin and expression the nalyzed ee eue b At or Akti by reduced were m of mTORC1/S6K which which mTORC1/S6K of m ccnb1 ) gene and protein geneand ) ether, these 6 DKO S6K Grb14 11

Page 12of54 Version postprint Page 13of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is diin whereas addition, compare cells hepatoma in 95% to 50% from decreased Interesti lines. cell hepatoma five and hepatocytes expressionGrb14is decreased inhepatoma human cel complex (Fig.7E). signalinsulinproliferative a stimulates IR the on the of release the that suggest data these However, activat the through homeostasis metabolic lipid and by Rb-E2F1 pathway. the tiigy nacd n l hptm cl lns Int lines. cell hepatoma all in enhanced strikingly compared first we transformation, hepatocyte by induced effect proliferative dependent re these Altogether, (Fig.7C-D). liver mice KO E2F1 ccne cycin increase the contrast, In signaling. insulin of absence the that indicating (Fig.7C), liver mice toconsecutive mice. KO E2F1 in Grb14i of effect the investigating Fig.S3A(supporting DKOmice S6K phosphorylationin Grb14 furthe was This (Fig.6E). mice Grb14i in activation Grb14 Fig.S3B Supporting (Fig.7A-B, immuno-labelling BrdU , ccna2 oneuain n liver on downregulation tmltd hshrlto o EK/, k ad S6K and Akt ERK1/2, of phosphorylation stimulated basal conditions,its insulinIn hepatic bindingto To investigate whether dysregulation of Grb14 expre Grb14 of dysregulation whether investigate To and Grb14 ccnb1 E2F1 Comment citer cedocument: downregulation was strongly reduced in E2F1 KOinE2F1downregulation strongly mic was reduced This article isprotected by copyright. All rights reserved. mRNA expression induced byinduced expression mRNA a hrl dtcal i piay eaoye, its hepatocytes, primary in detectable hardly was e2f1 xrsin n LRO ie Fg6) n o Rb on and (Fig.6F) mice iLIRKO in expression Hepatology Hepatology Grb14 GRB14 mediated by Akt-mTORC1 and the Rb-E2F1 the byand Akt-mTORC1 mediated lin A and B1 protein expression, and in and expression, protein B1 and A lin downregulation is mediated at least in part part in least at mediated is downregulation E2F1 expression did not modify upstream modify not did expression E2F1 erestingly, forced expression of of expression forced erestingly, ngly, The induction of hepatocyte proliferation hepatocyte of induction The endogenous repressive action of Grb14 of action repressive endogenous ilsrtd y h dcesd fet of effect decreased the by illustrated r ion of the Akt-mTORC1 pathway (1). (1). pathway Akt-mTORC1 the of ion mRNA level between freshly isolated freshly between level mRNA Grb14 ). As expected, the downregulation of of downregulation the expected, As ). glucoseIR theregulation promotes of ). We finally validated these results byresults these finally validated We ). l lines andHCC ut srnl sget ht h IR- the that suggest strongly sults GRB14 d to normal hepatocytes (Fig.8A). In In (Fig.8A). hepatocytes normal to d ssion could be related to human to related be could ssion downregulation was inhibited in in inhibitedwas downregulation similarly in E2F1 KO and WT WT and KO E2F1 in similarly expression was significantly significantly was expression

e, asby revealede, xrsin was expression Grb14 Ki67, 12 in

GRB14 Comment citer cedocument: This article isprotected by copyright. All rights reserved. expression is decreased in 60% of the human HCC sp HCC human the of 60% in decreased is expression GRB14 Grb14 oneuain oehr ih RA nuto i HCC in induction IR-A with together downregulation niiin Mroe, u suy lal identified clearly study our Moreover, inhibition. Hepatology Hepatology effect mostly dependent on IR expressionandIRon effect dependent mostly ation of protein and lipid synthesis, and cell andlipid protein of andcell synthesis, ation . The release of this proliferative effect in effect proliferative this of release The . o eeeae h lvr as fe partial after mass liver the regenerate to uig C dvlpet Oeal this Overall, development. HCC during fco o hptcts n highlights and hepatocytes on factor c ed in the liver, since the expression of expression the since liver, the in ed ie mtgnc cin s eitd by mediated is action mitogenic liver cial role in this particular situation, as it as situation, particular this in role cial n o te ellr otx (7 28). (27, context cellular the on ing r. Interestingly, this effect was likely likely was effect this Interestingly, r. Grb14 on in multiple cancers, and has been has and cancers, multiple in on hich dysregulation could be involved be could dysregulation hich peoeo, n urvld an unraveled and phenomenon, s ain ete SK r EP can 4EBP1 or S6K either vation, transcription factor. Our study also also study Our factor. transcription n HCC, playing a pivotal role in the in role pivotal a playing HCC, n oi (9 3) Hptcts are Hepatocytes 30). (29, nosis xrsin n os lvr is liver mouse in expression hepatocyte proliferation. proliferation. hepatocyte i sgaig ahas and pathways signaling lin ecimens the 14

Comment citer cedocument: This article isprotected by copyright. All rights reserved. Hepatology Hepatology but this is, to our knowledge, the first study first the knowledge, our to is, this but E2F1, E2F1 being pro-proliferative in early in pro-proliferative being E2F1 E2F1, and is considered as a potential marker for for marker potential a as considered is and carcinogenesis (35). E2F1 therefore arises therefore E2F1 (35). carcinogenesis rsin s nacd n ie fo obese, from liver in enhanced is pression lw el t etr pae 3) Hr we Here (34). phase S enter to cells llow proliferation. Overexpression of liver E2F1liver Overexpressionof proliferation. observed in liver regeneration after partial after regeneration liver in observed he downregulation of of downregulationhe s (36, 37). E2F1 is also overexpressed in overexpressed also is E2F1 37). (36, s ease S6K activation without modification without activation S6K ease ribute to liver oncogenesis by promoting promoting by oncogenesis liver to ribute hese studies, our data support a potent a support data our studies, hese ent kinases results in Rb inhibition and inhibition Rb in results kinases ent l division was triggered at day 2 while while 2 day at triggered was division l e (4. hs obe oe a nicely was role double This (34). ies wnstream IR signaling. The transient transient The signaling. IR wnstream rlfrto (1. n diin liver addition, In (31). proliferation counterregulatory mechanisms were mechanisms counterregulatory cular targets regulating the cell cycle cycle cell the regulating targets cular te ul ucin f 21 which E2F1, of function dual the y mitogens Many manner. dependent el ppoi (hs td, 8. This 38). study, (this apoptosis cell s eut i lvr ypai and dysplasia liver in results ns ly a ioa rl i cl cycle cell in role pivotal a plays Grb14 was mediated was 15 Page 16of54 Version postprint Page 17of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is diseases. pa and integrity liver of regulator important an as beremain clarified.to controllingXenopus loop feed-back negative a in cooperate (Checkpoin Chfr ligaseubiquitin and the Grb14 that s proliferative additional mediate partners binding effec mitogenic the consequently, and, signaling of feed- this by generated Grb14 in increase The (23). a hepatocytes primary in here shown as stimulation, inducti the by provided is hypothesis first A meal. re the of release the by stimulated is signaling IR feeding. The of question arisesthen the molecular and is proposed to play a role in the pathogenesis the in role a play to proposed is and methylation Chfr note, Of (40). cancers numerous in However, the molecular mechanisms involved in the d the in involved mechanisms molecular the However, consecu p62 of release the by altered be then could pro cell tumor and mitosis through cells of transit adapte the with complex constitutive a liver the in insulin signaling, and show that low level of of level low that show and signaling, insulin anti-pro an support data present Our (47). patients of expression high a and c cancer breast in progression cycle cell regulates or Grb14 of function mitogenic ver the on and available Grb14 of role metabolic the on focused were nui sgaig ahas ee iial activated similarly were pathways signaling Insulin oocytes (39). Chfr is a tumor-suppressor gene, whogene,tumor-suppressor a is Chfr (39).oocytes Comment citer cedocument: This article isprotected by copyright. All rights reserved. GRB14 is an independent good prognosis factor for breast for factor prognosis good independent an is Hepatology Hepatology GRB14 Grb14 mechanism that triggers cellmechanism that division when liferative function of Grb14 downstream of downstream Grb14 of function liferative hpyilgcleouin f hoi liver chronic of evolution thophysiological ignals. Interestingly, we recently showed showed recently we Interestingly, ignals. ells in an insulin-dependent manner (46), (46), manner insulin-dependent an in ells t with forkhead and Ring Finger domains)FingerRing and withforkhead t ieain 4-5. el yl progression cycle Cell (43-45). liferation rsie cin f r1 bt o atr a after not but Grb14 of action pressive of this cancer (41, 42). Grb14 also forms forms also Grb14 42). (41, cancer this of p2ssm, hc cnrl te timely the controls which p62/sqstm1, r t of insulin. An alternative is that Grb14 that is alternative An insulin. of t back loop can tightly regulate the length the regulate tightly can loop back correlates with the progression of HCC of progression the with correlates n f r1 epeso udr insulin under expression Grb14 of on could be a be could ie to tive d rvosy eotd n adipocytes in reported previously nd ecreased nui-nue cl poieain in proliferation cell insulin-induced xrsin n acr (14). cancer in expression fw nomtos r currently are informations few y by se promoter is hypermethylatedis promoter se Grb14 Grb14 GRB14 bona fide bona iecn. ot studies Most silencing. oneuain r by or downregulation expression in HCC in expression marker of HCC. of marker cancer GRB14 16

Among the molecular mechanisms potentially underlyi potentially mechanisms molecular the Among Comment citer cedocument: This article isprotected by copyright. All rights reserved. Hepatology Hepatology e is to date no evidence for an implication of implication an for evidence no date to is e tiation, but our study provides evidence that that evidence provides study our but tiation, ulinemiaincreased and HCCprevalence. lin axis deserves a particular interest (2). interest particular a deserves axis lin istance and elevated levels of circulating of levels elevated and istance eain n ie, n ofr n attractive an offer and liver, in feration itn t te cin f nui, uh as such insulin, of action the to sistant stv t te omn, uh as such hormone, the to nsitive ng the link between metabolic between link the ng 17 Page 18of54 Version postprint Page 19of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is Genom’ic and Dumont Florent thank to like would authors The ACKNOWLEDGMENTS critical reading of the manuscript. manuscript. the of reading critical ackno gratefully are Gilgenkrantz Hélène and Perret kindly for Panzyuk Ganna and Pende Mario analyses, analysis statistical for Chrétien Yves samples, HCC Hosp Groupe the from »Est Tumeur« the acknowledge Pari Bernard, Claude Institut the from Biochemistry performi for Paris) U1016, Inserm Cochin, (Institut ani Cochin’s the of staff the and Lagoutte Isabelle Département Hospitalo-Universitaire AUToimmun(DHU) France. de Ile Région the of help the with equipped

Isiu Cci, nem 11, Paris) U1016, Inserm Cochin, (Institut Comment citer cedocument: This article isprotected by copyright. All rights reserved. Mice used in this study were housed in an animal fa animal an in housed were study this in used Mice Hepatology Hepatology o mcora eprmns n analysis, and experiments microarray for , Dominique Wendum for histopathology for Wendum Dominique , a fclt, h Cci HsI Facility HistIM Cochin the facility, mal Franck Letourneur from the Plateform the from Letourneur Franck ng liver sections, the Plateforme of of Plateforme the sections, liver ng s, for plasma analysis. The authors authors The analysis. plasma for s, h wr ws efre wti the within performed was work The lde fr epu dsusos and discussions helpful for wledged provinding S6K DKO mice. Christine mice. DKO S6K provinding e and HORmonaldiseasesS. italier HUEP of AP-HP for access to access for AP-HP of HUEP italier

cility 18 Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is rece Insulin CR. Kahn A, Kleinridders J, Boucher 9. Scha P, Meyts de R, Borup J, Palsgaard M, Jensen 8. Vign L, Sciacca G, Pandini F, Frasca A, Belfiore 7. D,C L, Wendum L,AoudjehaneFartoux H, Chettouh 6. E Piura RH, Bell M, Zakikhani ME, Gleave ME, Cox 5. De C, Vigouroux L, Fartoux Lequoy M, H, Chettouh 4. T Diabetes: and Obesity D. LeRoith EJ, Gallagher 3. D.The LeRoith Gallagher EJ, role proliferating 2. the and signalling Insulin CR. Kahn AR, Saltiel 1. REFERENCES 10. Svegliati-Baroni G, Ridolfi F, Di Sario A, Casi A, Sario Di F, Ridolfi G, Svegliati-Baroni 10. in IR-transfected L6Biochem J 2008;412: myoblasts. ha peptide synthetic a and insulin by (IR) receptor Rev2009;30:586-623.Endocr recepto factor growth receptor/insulin-like insulin dysregulationEGFR-mediated RNA of splicing factors hu in overexpressed is receptor-A insulin Mitogenic receptorInsulinexpressionby cance prostate human carcinoma. Liver 2015;35:2203-2217.Int the and pathogenesis the in signalling insulin and Mortality.Cancer-Related Physiol 2015;95:727-7Rev Endocrinolcancer. Trends 2010;21:610-618. Metab Naturemetabolism.2001;414:799-806. accumulation by human hepatic stellate cells: diffe cells: stellate hepatic human by accumulation sti factor-1 growth insulin-like and Insulin al. et states.resistant ColdHarb Perspect SpringBiol 20 Comment citer cedocument: This article isprotected by copyright. All rights reserved. Hepatology Hepatology ni A, Marucci L, Gaggiotti G, Orlandoni P, Orlandoni G, Gaggiotti L, Marucci A, ni of insulinof insulin-like growth in and factors uae rlfrto ad ye collagen I type and proliferation mulate hbis n hsooy n disease. and physiology in hybrids r s different effects on gene expression gene on effects different s 14;6. tr inln i nra ad insulin- and normal in signaling ptor man hepatocellular carcinoma due to due carcinoma hepatocellular man r R Isln eetr sfrs and isoforms receptor Insulin R. eri sbois-Mouthon C. Hyperinsulinaemia C. sbois-Mouthon rential effects on signal transduction signal on effects rential 435-445. Prostaters. 2009;69:33-40. laperon A, Chretien Y, Rey C, et al.et C, ReyY, Chretien A,laperon e nrae Rs o Cne and Cancer of Risk Increased he 48. , Vickers E, Cunningham M, et al. et M, Cunningham E, Vickers , . Cancer. Res 2013;73:3974-3986. euain f lcs ad lipid and glucose of regulation lncl ore f hepatocellular of course clinical fr . ciain f h insulin the of Activation L. ffer 19 Page 20of54 Version postprint Page 21of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is 20. Nemazanyy I, Montagnac G, Russell RC, Morzyglod RC, Russell G, Montagnac I, Nemazanyy 20. Browae K, Cailliau N, Carré C, Hampe D, Goenaga 19. 18. Carré N, Caüzac M, Girard J, Burnol A-F. Dual e Dual A-F. Burnol J, Girard M, Caüzac N, Carré 18. J, Girard F, Authier V, Bereziat B, Desbuquois 17. Cariou D, Perdereau A, Kasus-Jacobi V, Bereziat 16. E Clauser C, Auzan D, Perdereau A, Kasus-Jacobi 15. of Regulation AF. Burnol N, Carre B, Desbuquois 14.

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Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is S Mukherjee VK, Mootha P, Tamayo A, Subramanian 48. Chen Z,J, Xie Jiang ZhangX, M, Huang Wu O, X, 47. Mus A, Swarbrick RJ, Lyons J, Parmar R, Kairouz 46. M MT, Diaz-Meco K, Greis R, Amanchy JF, Linares 45. 44. Browaeys-Pol K, Cailliau D, Perdereau B, Cariou 43. hy Promoter S. Li T, Hao J, Yang H, Zhang Z, Li 42. S, Inoue S, Nomoto N, Kanazumi K, Hibi M, Sakai 41. Brande KM, Smits V, Melotte AH, Cleven S, Derks 40. Jpn J ClinJpn Oncol 2013;43:1064-1072. cancerpatientsbreastgood prognosis factor tr for breastcancer human J cells. Cell Physiol 2005;203: ro its and modulator signal Grb14 the of regulation Cell Biol 2011;31:105-117. m through cells of transit timely the controls cdk1 andLiver LipogenesisResistance.Selective Insulin and Insulin between Talk Cross Grb14-Mediated Novel L Morzyglod L, Popineau insulinsignaling Kinaseby Protein Cz. recruiting Grb1binds ZIPprotein adapterThe al. et J, Girard hepatocell genesresponse in hepatocellularcarcinoma. Cell Bi advanced in gene CHFR Hepatogastroenterology2005;52:1854-1857. the of methylation CancerMetastasismedicine. Rev 2014;33:161-171. f biomarker: cancer a as CHFR for evidence Emerging genome-wideNatl expression profiles. Proc Acad Sci knowledge a analysis: enrichment set Gene al. et A, Comment citer cedocument: This article isprotected by copyright. All rights reserved. , Carré N, Cauzac M, Bossard P, Prip-Buus C,Prip-BuusLenoirCauzacP, BossardN,M, Carré , Hepatology Hepatology MolCellBiol 2002;22:6959-6970. eated withneoadjuvant chemotherapy.eated Liu H, et al. Grb14 as anet as Grb14 independent LiuH, al. itosis and tumor cell proliferation. Mol proliferation. cell tumor and itosis 4 and regulates its inhibitory action on inhibitory its action regulates and 4 s JC, Azad N, van Criekinge W, et al. et W, Criekinge van N, Azad JC, s E Brza V VserCge M, Vasseur-Cognet V, Béréziat E, y MolCell Biol 2016;36:2168-2181. le in cell cycle progression of MCF-7 of progression cycle cell in le 85-93. ol Int2012;36:427-432.ol oscat J. Phosphorylation of p62 by p62 of Phosphorylation J. oscat U S 2005;102:15545-15550. A U , Ebert BL, Gillette MA, Paulovich MA, Gillette BL, Ebert , permethylation of DNA damage damage DNA of permethylation bsd prah o interpreting for approach -based aea , aa A Aberrant A. Nakao S, Takeda rv E, ay J Hormonal RJ. Daly EA, grove o tmr ilg t precision to biology tumor rom 6/r2 ahas Regulates Pathways p62/Nrf2 lr carcinoma. ular V, etal.V,

23 Page 24of54 Version postprint Page 25of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is gene sets. All results are shown in the Supporting Supporting the in shown are results All sets. gene GSE813 (GEO (n=3) Grb14i or (n=3) USi with injected with performed were (48) (GSEA) analysis enrichment f lysates a liver in blot pathways signaling Western intracellular (B) part). (right expression protein are Results expression. cyclins of analysis qRT-PCR of analysis stimulat and (t=0h) plating after adenovirus Grb14i Prima challenge. mitotic a to submitted hepatocytes : 2 Figure GAPDHas loadingis used control. representativ three analysis, blot Western For 18S. mice. USi to compared Grb14i for **p<0.01 *p<0.05, are Results presentation. of clarity for panels two GAPD same the (with experiments same the from are F blotqRT-PCRandWestern analysis (left) (right) of qu and (x20) sections l liver of KEGG immunohistochemistry the with mice Grb14i from liver in regulated si a showing pathway enriched top the for plot GSEA N NES, 0.001). < value q (FDR shown are enrichments of analysis qRT-PCR (A) days. shRNA (Grb14i) Grb14 or (USi) unspecific expressing wer mice C57Bl/6J mice. in proliferation of hepatocyte inhibition Liver-specific : 1 Figure FIGURE LEGENDS Ki67 Grb14 expression. (B) Western blot analysis of Grb14 and Grb14 of analysis blot Western (B) expression. onrglto ehne cl division cell enhances down-regulation Comment citer cedocument: This article isprotected by copyright. All rights reserved. Grb14 xrsin lf pr) n qatfcto o Grb14 of quantification and part) (left expression Hepatology Hepatology Grb14 mrvs nui sgaig n induces and signaling insulin improves o Ui n Gb4 mc. C Gn set Gene (C) mice. Grb14i and USi rom cell and in cycleB blots Western markers. ed with the mitogenic mix. (A) qRT-PCR (A) mix. mitogenic the with ed ry hepatocytes were infected with USi or USi with infected were hepatocytes ry e intravenously injected with adenovirus adenovirus with injected intravenously e h ma ± E ( = pr group). per 6 = (n SEM ± mean the the mean ± SEM of three independent three of SEM± mean the mRNA levels are expressed relative to to relative expressed are levels mRNA e samples (from six) are shown and and shown are six) (from samples e niiain f rU oiie el. (F) cells. positive BrdU of antification trancriptomic data from C57Bl/6 mice mice C57Bl/6 from data trancriptomic n lvr wr hretd fe two after harvested were livers and ayi o Gb4 xrsin and expression Grb14 of nalysis al S ad ee ny h best the only here and S1 Table H loading control) and are shown in shown are and control) loading H nfcn ercmn fr ee up- genes for enrichment gnificant ist of cell cycle genes. (E) BrdU BrdU (E) genes. cycle cell of ist 7 ad ih iCra n KEGG and BioCarta with and 87) raie Ercmn Soe (D) Score. Enrichment ormalized n vitro in cyclins expression. (C) expression. cyclins n rmr mouse primary in 24

Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is Figure 4 : Hepatocyte division upon division Hepatocyte : 4 Figure loading control. per 8-10 from samples representative twoanalysis, ***p<0.005 for Grb14i compared to USi mice. mice. USi to compared 10 Grb14i to for 8 ***p<0.005 = (n SEM ± mean the are Results expression. qRT (E) expression. cyclins and activation pathways (D cells. positive BrdU of quantification and (x20) ( of analysis blot Western expression. IR of analysis qRT-PCR (A) infection. adenovirus. Grb14i or USi with infection the before mice. USi to compared Grb14i for ***p<0.005 = (n SEM ± mean the are Results expression. cyclins b original the of cropping indicates line black the Samples expression. cyclins and pathways signaling Western (B) cells. positive BrdU of quantification immunohistochemistr BrdU (A) 4.5mg/kg). rapamycin: or Gr inhibitors indicated or the USi with intraperitoneally with infected were mice C57Bl/6J pathway. IR littermate proliferation hepatocyte Grb14i-induced : 3 Figure levels expressedrelative are 18S. to Grb14i for ***p<0.005 **p<0.01, *p<0.05, cultures. GAPDHas loadingis used control. ofblotanalysis,blotsWestern tworepresentative a levels mRNA mice. Grb14i DMSO-treated to compared oprd o IR to compared lox/lox lox/lox oto mc wr ijce wt tmxfn o delet to tamoxifen with injected were mice control Comment citer cedocument: ie mN lvl ae xrse rltv t 1S F 18S. to relative expressed are levels mRNA mice. This article isprotected by copyright. All rights reserved. Grb14 expression. (C) BrdU immunohistochemistry of liver of immunohistochemistry BrdU (C) expression. Grb14 Hepatology Hepatology downregulation is mediated by the Akt/mTORC1 the by mediated is downregulation # samples from 5-6 per groupshown5-6areand per samples from p<0.05, p<0.05, # ) Western blot analysis of insulin signaling insulin of analysis blot Western ) blot analysis of Grb14 expression, insulin expression, Grb14 of analysis blot p<0.05, h cnrl MO At VI 50mg/kg, VIII : (Akti DMSO control the os () R-C aayi of analysis qRT-PCR (C) lots. Livers were harvested after two days of days two after harvested were Livers group are shown and GAPDH is used as used is GAPDHand shown are group compared to USi hepatocytes. mRNA mRNA hepatocytes. USi to compared were analyzed on the same blots and blots same the on analyzed were s eitd y h I. LRO and iLIRKO IR. the by mediated is -PCR analysis of of analysis -PCR ) R-C ad uniiain of quantification and qRT-PCR B) - pr ru) *<.5 **p<0.01, *p<0.05, group). per 5-6 ## e xrse rltv t 1S For 18S. to relative expressed re o lvr etos x0 and (x20) sections liver of y ## e gop. p00, **p<0.01, *p<0.05, group). per p<0.01, 1i dnvrs n injected and adenovirus b14i p<0.01, ### lvr R he weeks three IR liver e ### p<0.005 for inhibitor- inhibitor- for p<0.005 <.0 fr iLIRKO for p<0.005 Ki67 r etr blot Western or n cyclins and Ki67 sections and 25 Page 26of54 Version postprint Page 27of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is expre E2F1 mRNA (F) indicated. as rapamycinor Akti expression E2F1of (lowerpart) inUSi liver from a expressi E2F1 of and phosphorylation and expression Score. Enrichment Normalized NES, mice. Grb14i from gen with (25)) GSE19004 (GEO mice in genes Rb three dow and up genes of enrichment significant a showed ( triplicates. in performed experiments independent Resul Grb14i. or USi with treated hepatocytes mouse lucifer E2F-RE an of Activity (C) control). loading s experiment same the from are (B) in Blots Grb14i. culture primary in (B) and mice Grb14i and USi from ex Rb of analysis blot Western (A) pathway. Rb/E2F1 # ***p**p<0.01, *p<0.05, group). per 4 to 3 = (n SEM of analysis qRT-PCR (D) expression. i of analysis blot Western (C) expression. Grb14 of qRT-PCR immunohistochemis (B) cells.positive BrdU of quantification BrdU (A) days. two after analyzed adeno Grb14i or USi with injected were mice control hepato Grb14i-induced in involved is S6K : 5 Figure Figure 6 : Hepatocyte proliferation induced by live by induced proliferation Hepatocyte : 6 Figure groupper shown are and GAPDH asloadingis used co analysi blot Western For 18S. to relative expressed iLIRKOandinjected mice withGrb14iadenoviUSi or p<0.05, ## p<0.01, ### Comment citer cedocument: p<0.005 for S6K DKO compared to WT Grb14i mice. mRN mice. Grb14i WT to compared DKO S6K for p<0.005 This article isprotected by copyright. All rights reserved. Ki67 Hepatology Hepatology and cyclins expression. Results are the mean ± mean the are Results expression. cyclins and nd Grb14i mice nd Grb14i treated control DMSO,with mice r r and quantification of Western blot analysisblot of Western quantification and s gn rpre tasetd n primary in transfected reporter gene ase D) Gene set enrichment analysis (GSEA) (GSEA) analysis enrichment set Gene D) Grb14 <0.005 for Grb14i compared to USi mice. to compared Grb14i for <0.005 s, two representative samples from 3-4 from samples representative two s, rus. hown in Fig.2B (with the same GAPDH same the (with Fig.2B in hown nsulin signaling pathways and cyclins cyclins and pathways signaling nsulin ssion measured by qRT-PCR in IR qRT-PCRin by ssion measured on (upper part) and qRT-PCR analysis analysis qRT-PCRand part) (upper on cyte proliferation. S6K DKO and WT WT and DKO S6K proliferation. cyte nregulated by liver invalidation of the of invalidation liver by nregulated pression and phosphorylation in liver liver in phosphorylation and pression iu ad ies ee avse and harvested were livers and virus ntrol. hptcts rae wt Ui or USi with treated hepatocytes d e expression profile induced in liver in induced profile expression e s r te en SM f three of SEM ± mean the are ts downregulation is mediated by the by mediated is downregulation E Wsen lt nlss f Rb of analysis blot Western (E) r o lvr etos x0 and (x20) sections liver of try A levels are levels A lox/lox 26

Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is Figure 8 : Figure inhibition insulinsignaling enhances promotesand of control the to contributing activity, catalytic sign IR of Grb14 by control the through homeostasis (E) control.loading as used GAPDHis andshown are rep two analysis, blot Western For 18S. to relative of ## analysis blot Western of quantification and qRT-PCR qu and (x20) sections liver of immunohistochemistry proliferation hepatocyte Grb14i-induced 7 : Figure tumor liver(NT)tissuesamples. adjacent of analysis PCR G of expression increased the showing exp blots Western independent three of SEM ± mean the are Results i or presence the in cultured and (EV) vector empty pcDN the with transiently transfected cells Huh6 or after number Cell (B) HH. to compared when p<0.05 * Resultlines. cell in hepatoma and hepatocytes(HH) f ***p<0.005 **p<0.01, *p<0.05, group). per 13 to 8 Ki67 G USior injectedwith littermates control and mice cyclins and pathways signaling insulin of analysis p<0.01, GRB14 and cyclins expression from USi- andcyclins expression Grb14i-miceli or from ### lf pr) and part) (left GRB14 <.0 fr 21 O oprd o T ie mN level mRNA mice. WT to compared KO E2F1 for p<0.005 GRB14 status in human hepatoma cells and HCC tumors. (A) tumors. HCC and cells hepatoma human in status Comment citer cedocument: This article isprotected by copyright. All rights reserved. (C) and (C) E2F1 rgt at epeso i nra fehy isolated freshly normal in expression part) (right E2F1 (D) mRNA expression in 85 paired HCC (T) and non and (T) HCC paired 85 in expression mRNA (D) Hepatology Hepatology liver metabolic homeostasis. Right part : Grb14 Grb14 : part Righthomeostasis. livermetabolic rb14i adenovirus. (D) qRT-PCR analysis ofqRT-PCR(D) analysisadenovirus. rb14i A3-Grb14 expression vector or the control the or vector expression A3-Grb14 a mTORC1-Rb/E2F1 proliferativea signal. mTORC1-Rb/E2F1 expression in liver lysates from E2F1 KO E2F1 from lysates liver in expression resentative samples from 8-13 per group per 8-13 from samples resentative s niie i EF K mc. A BrdU (A) mice. KO E2F1 in inhibited is s are the mean ± SEM (n = 4 per group),per 4 ± = the SEM(n mean are s of of te bec o isln s indicated. as insulin of absence the n r r1i oprd o USi. to compared Grb14i or Model for the regulation of hepatocyte of regulation the Modelfor niiain f rU oiie el. (B) cells. positive BrdU of antification 60h of culture for PLC/PRF5, HepG2 PLC/PRF5, for culture of 60h ver. Resultsver. ± the are mean SEM(n = ln. et at: r1 rpess IR represses Grb14 part : Left aling. b4 n rnfce cls (-) qRT- (C-D) cells. transfected in rb14 Grb14 eriments, *p<0.05. Upper part : part Upper *p<0.05. eriments, expression. (C) Western blot Western (C) expression. qRT-PCR analysis analysis qRT-PCR ae expressed are s # human p<0.05, 27 Page 28of54 Version postprint Page 29of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is USi (n=3) or Grb14i (n=3) (GEO GSE81387) (n=3) GSE81387) withor USi B Grb14i and (n=3) (GEO Figure1: Liver- immunohistochemistryliver of sections qu(x20) and and qRT- and Normalized Enrichment Score. (D) GSEA plotNormalized forGSEA the (D) Score. Enrichment (with the same GAPDH loading GAPDH (withshown control)are same the and enrichment analysis (GSEA) enrichment analysis (48)were withperformed (Grb14i)livers two and after harvested shRNA were expression and intracellular signaling pathways in expression intracellularsignaling and pathways mice.C57Bl/6J mice injected were intravenously wit the Supporting Table S1 and hereSupportingS1 the Table only bestthe enri part) and quantification of Grb14 of quantification part)and protein expressio forgenes up-regulated fromGrb14i in miceliver wi PCR (right) PCR analysis cell of cycle Western markers. specific inhibition of Grb14 improves insulin signainhibition specific Grb14 insulin of improves Comment citer cedocument: This article isprotected by copyright. All rights reserved. Hepatology Hepatology antification of BrdU positive BrdU of (F)antification Western cells. bl liver lysates from USi liverfromUSi Grb14i and mice. Gene lysates (C) se top showing a enrichedsignificant enrichmepathway

days. (A) qRT-PCR days.qRT-PCR Grb14(A) of analysis expression (lef

n (right part). (B) Western(B) part). blot analysis Grb14 of (right n h adenovirus expressing h (USi) or Grb14 unspecific in panels fortwo clarity presentation.Results of trancriptomic data fromC57Bl/6 data mice injected trancriptomic with chments are shown (FDR q value (FDR shown are 0.001).NES, < chments ioCarta gene KEGG sets. and All i results shown are th the KEGG list KEGG the cell of th (E) BrdU cycle genes. blots in B and F are fromare F experiments blots and same the in B lingproliferationinduces hepatocyte and in

ot (left) ot are t t t t nt nt

n n Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

expressed relative to 18S. For expressed18S. to Western relative blot analysi the mean ± SEM (n = 6per(n= SEM ± mean the group).**p<0.01*p<0.05, Comment citer cedocument: This article isprotected by copyright. All rights reserved. GAPDH is used as loading GAPDH control. 191x244mm(300 300 x DPI) Hepatology Hepatology s, three s, re

for Grb14i levels mice. mRNA USi compared to are presentative samples (from six) are shown and and six) presentativeshown are samples (from Page 30of54 Version postprint Page 31of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

of Grb14 and cyclins Grb14 of expression. and qRT- (C) Figure2: Grb14 down- and stimulated with mitogenicthe and qRT-PCR mix.(A) a mitotic were a challenge. hepatocytes Primary infec three**p<0.01,***pindependent*p<0.05, cultures. regulation enhances cell enhances division vitro in prima in regulation Comment citer cedocument: This article isprotected by copyright. All rights reserved. mRNA levels mRNA relative expressedare 18S. to 191x212mm(300 300 x DPI) PCR analysis cyclins PCR of expression.the are Results Hepatology Hepatology ted with USi or Grb14i adenovirus after plating tedorafter Grb14iwith (t= USi adenovirus

analysisKi67 Westernof blot expression. analy (B)

<0.005 for Grb14i compared to USi hepatocytes. <0.005for Grb14i hepatocytes. USi compared to ry mouse hepatocytes submitted hepatocytes to ry mouse mean ± SEM of of ± mean SEM

0h) sis Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is Grb14iLivers wereadenovirus. two after harvested cyclins expression. Results are the mean ± SEM (n= SEM ± cyclinsmean expression.the Results are analysis of insulin of analysis signaling a activation pathways Figure 3 : Grb14i-induced hepatocyte proliferation Figure3: Grb14i-induced hepatocyte mice. mRNA levels mice.mRNA relativeFo expressed are 18S. to forGrb14i mice. ##p<0.01, #p<0.05, USi compared to controldelete to miceinjectedwith tamoxifen were immunohistochemistryliver of sections qu(x20) and (B) qRT-PCR and quantification and qRT-PCR (B) Western of blot anal from8-10is GAPDH usedper as and group shown are Comment citer cedocument: This article isprotected by copyright. All rights reserved. 187x234mm(300 300 x DPI) Hepatology Hepatology nd cyclinsnd Ki67 of analysis expression. (E) qRT-PCR daysinfection. qRT-of (A) is mediated by the IR. iLIRKO iLIRKO andIR. littermatebythe ismediated IRlox/ liver IR three weeks before liver IR infectionthe with USi 8to 10 per group). **p<0.01,***p<0.005*p<0.05,

rWesternrepresentative two blot analysis, samples antification of BrdU positive BrdU of Western(D) antification blcells. ###p<0.005 for iLIRKO compared###p<0.005for iLIRKO IRlox/lox to ysisGrb14 of expression. BrdU (C) loadingcontrol. PCR expression. PCR of analysis IR

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Page 32of54 Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is Grb14i###mice. ##p<0.01, #p<0.05, USi compared to C57Bl/6Jmice orwere Grb14iwith USi infected aden analysis of Grb14 of expression, analysis insulin signaling pat cyclins expression. Results are the mean ± SEM (n= SEM ± cyclinsmean expression.the Results are the same blots and the blackthe blots and same the line indicates croppin Grb14ilevelsexpressed are mice. mRNA to relative immunohistochemistryliver of sections qu(x20) and Figure 4 : Hepatocyte division upon Grb14 upon downregulFigure 4: Hepatocyte division inhibitors or the control DMSO (Akti VIII : 50mg/kg (Akti VIII inhibitorsor controlthe DMSO two samples from5-6two GAPDH per and group shown are Comment citer cedocument: This article isprotected by copyright. All rights reserved. 189x270mm(300 300 x DPI) Hepatology Hepatology hways and cyclins expression. and Samples hways were analyzed g of the originalthe g Kiof of analysis blots.qRT-PCR (C) 18S. For Western For blot analysis, 18S. representativeblot ovirus and injected and intraperitoneally ovirus with indithe 5-6 per group). **p<0.01,***p<0.005*p<0.05, for

antification of BrdU positive BrdU of Western(B) antification blcells. ation is ation mediatedpathway. Akt/mTORC1 bythe p<0.005for inhibitor- DMSO- to compared , rapamycin , : 4.5mg/kg).BrdU (A) is used as loading isusedcontrol. as

67 and 67and treated cated cated s of of s ot ot on on Page 34of54 Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

pathways cyclins expression. and analyspathways qRT-PCR (D) #p<0.05, ##p<0.01, ###p<0.005 ##p<0.01, #p<0.05, DKO comparedfor S6K Figureis involved5: Grb14i-induced S6K in hepato relative to 18S. For Westernrep relativeFor two blot analysis, 18S. to quantification of Western of Grb14 quantification blotof ex analysis injectedlivers adenovirusor and Grb14i with USi w immunohistochemistryliver of sections qu(x20) and ± SEM (n= 4per 3to SEM ± group). **p<0.01,***p<0.05, Comment citer cedocument: This article isprotected by copyright. All rights reserved. GAPDH is used as loading GAPDH control. 187x201mm(300 300 x DPI) Hepatology Hepatology resentativesamples from3-4 per an group shown are pression.Western (C) insulin blotof analysis sign cyte proliferation. S6K DKO DKO and proliferation.S6K control miceWT wer cyte ere harvested and analyzed and harvested (A) after eredays. BrdU two isKi67 cyclinsof exp and

antification of BrdU positive BrdU of qRT-PCR (B) antification cells. an *p<0.005for Grb14i mice. USi compared to to WT Grb14i mice. mRNA levels Grb14i WT to expressedare mice. mRNA ression. Results are the mean Results mean the are ression.

aling d d e

Page 36of54 Version postprint Page 37of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is genereporter primary hepatocy mouse in transfected pathway. (A) Western blot pathway. analysis Rb of expression expression (upper part) and qRT-PCR analysis expressionqRT-PCR E2F of (upper part)and Normalized(E) Western Score. blotEnrichment analy showed a significant dowenrichment showed a genes of and up SEM of three of SEM independent inperformed experiments t mice treated with control DMSO, or micerapamycin Akti treated a with control DMSO, experimentloa GAPDH (with in same Fig.2B the shown Figure6: Hepatocyte proliferation induced bylive in mice (GEO GSE19004 (25)) inmice GSE19004 with gene(GEO pr expression and (B) in(B) primary and cultured treated wit hepatocytes qRT-PCR in IRlox/lox and iLIRKO in and IRlox/lox qRT-PCR mice injected with Comment citer cedocument: This article isprotected by copyright. All rights reserved. Hepatology Hepatology and phosphorylation in liver from USi and Grb14i phosphorylation inand liverand m USi from r Grb14 downregulation Rb/E2F1bythe ris Grb14 mediated tesortreated Grb14i.with USi Resu 1expression Grb (lowerpart)inand liver fromUSi

sisRb of E2F expressionof phosphorylation and and

s indicated. s expression mRNA (F) E2F1 measuredby nregulatedthreethe of Rb byliver invalidation ge riplicates. (D) Gene(D) (GSEA) riplicates. setenrichment analysis h USi or Grb14i. Blots in (B) are from the same USi same h fromthe or Grb14i. in are Blots (B) dingan control).of luciferase Activity E2F-RE (C) ofile induced in liver from Grb14i mice. NES, ofileinmice.liver fromGrb14i NES, induced USi or Grb14i adenovirus. or Grb14i USi adenovirus. lts are the mean ± ltsmean the are

nes 14i ice

Version postprint Page 39of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is through the control by Grb14 of IR signaling. controlLeftthe through byGrb14 IR of pathways cyclins expression and pathways in liverfr lysates compared to USi. #p<0.05, #p<0.05, USi. compared to ###p<0.005 ##p<0.01, for orGrb14i analysis qRT-PCR (D) Ki67adenovirus. of expressed relative to 18S. For expressed18S. to Western relative blot analysi quantification of Western of Grb14 quantification blotof ex analysis immunohistochemistryliver of sections qu(x20) and the control of liver metabolic homeostasis. Right homeostasis. p controlthe liver of metabolic shown and GAPDH and isshown used as loading control. (E) Mod Results are the mean ± SEM± Resultsmean the 13are per 8to (n= group). Figure 7 : Grb14i-induced hepatocyte proliferation Figure7: Grb14i-induced hepatocyte Comment citer cedocument: This article isprotected by copyright. All rights reserved. promotes a mTORC1-Rb/E2F1 promotesa proliferative signal. Hepatology Hepatology om E2F1 KO mice and controlmice KO littermates and E2F1 om injected wi part : part c activity, Grb14catalytic represses IR s, two representative8-13 two per s, groupsamples from a pression.Western (C) insulin blotof analysis sign art : art Grb14insulin inhibition enhances signaling a and cyclins expression and or fromUSi- Grb14i-mice liv

antification of BrdU positive BrdU of qRT-PCR (B) antification cells. an el for the regulation of hepatocyte homeostasis forregulationthe homeostasis elhepatocyte of *p<0.05, **p<0.01,***p<0.005*p<0.05, for Grb14i E2F1 KO compared to WT WT compared to KO levels mice.E2F1 mRNA are is inhibited in E2F1 KO mice.inhibited KO (A) inE2F1 is BrdU

ontributing to aling th USi USi th nd er. d re Version postprint Page 41of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

cell4per(n= SEM ± Results g mean lines. the are expressionvector or controlthe empty vector (EV) part) and E2F1 (right E2F1 part) part)and expressionin fr normal GRB14 (C) and E2F1 and mRNA (D) (C) GRB14 expression in 85 paired Westernexpression blots showing increased the G of Figure 8 : GRB14 status in humanhepatoma cellsFigure 8:status GRB14 and after 60h of culture of 60h forafter PLC/PRF5, HepG2 or Huh6ce as indicated. Results are the mean ± SEM of three indicated.of SEM ± i Results as mean the are Comment citer cedocument: This article isprotected by copyright. All rights reserved. 187x259mm(300 300 x DPI) Hepatology Hepatology samples. samples. eshly isolated human hepatocytes isolated (HH) human in eshly hepato and roup), and cultured and i of in in absence presencethe the or

rb14 incells.analysis transfected qRT-PCR rb14 o (C-D) lls transientlywith pcDNA3-Grb14 the transfected ndependentUpper :experiments, part *p<0.05. HCC tumors. qRT-PCR (A) analysis (left GRB14 of HCC (T) tumor non HCC and (NT) liver adjacent tissue * p<0.05 when compared*p<0.05 when HH. to Cell(B) number

nsulin ma ma f f Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is controlled for mycoplasma contamination. contamination. mycoplasma for controlled shortATCC tandem repeats. Cell lines were cultured as previously reported France)Cochin, Huh (ATCC). Collection Hepatoma cells and culture transient transfection experiments of cultures humanPrimary hepatocytes were established previously as described measured measured according(LifeTechnologies) Luciferase toinstructions. theactivity manufacturer’s was recommendations. Serum recommendations. or TransIT Scientific) transfected withtransfected empty empty withwere transfected 0.5 mitogenic factors mitogenic pyruvatesodium 0.2mM) and3pfu/cell of USiMedium adenovirus. or Grb14i hepatocytes cultures Primary PROCEDURES EXPERIMENTAL Supporting Protein Protein 60 hand counted using Bio spreading, hepatocytes were spreading, bymice an HepG2, Hep3B, and HuH7 cells were obtained Americanfrom the Type Culture Hepatocytes were isolated from livers were eight ofHepatocytes isolated from fed pcDNA3 pcDNA3 extraction and immunoblotting every 12h

informations in situ . plasmid by using Lipofectamine 3000 (Huh6, PLC/PRF5, Thermo Fisher

Cell linewas authentication routinely by using performed apanel of nine was was a

from 12 to 60 to 12 from collagenase method as described previously 3X E2F1 3X freshly replaced freshly This article isprotected by copyright. All rights reserved. Comment citer cedocument: - 6 andPLC/PRF5 providedcells were by Christine Dr Perret (Institut 2020 (HepG2, Mirus Bio according LLC) to manufacturers - m

deprived cells were then treated or not withdeprived or were insulin treated (10 then cells - g of pcDNA3g the coding plasmid rat for Grb14 (5) Rad TC20 cell counter.

incubated and luciferasereporter assays - RE luciferase plasmid luciferase RE

afte

with with r reporter transfection reporter r at 12h andat 36h. Hepatology Hepatology Cells inseeded 24 mitogenic mitogenic (EGF factors 50ng/ml

Primary cultured hepatocytes Primarywere cultured hepatocytes

(2) to ten to - .

well (3 dishes

using - week

(1) - lipofectamine 2000lipofectamine old male C57BL6/J . Four hours after

- , insulin, 0.68 (4) 5x10

or of a control acontrol of or c

ontaining the the ontaining and routinely and routinely

- (3) 4 8

cells/well) cells/well) M) during M) during .

m M, M, Page 42of54 Version postprint Page 43of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is expressed as as expressed relative mRNA andwere normalized to 18S. using a SYBRperformed PCR Green Master Mix data Diagnostic). were (Roche The primer and(dT)15 primer random (Promega). real Quantitative using Superscript transcription reverse II reverse tr Rb(S7 quantity o quantity mRNA contentHPRT and same was calibrator. the relatively expressed to each For primers. expression sample, waschemistry andspecific that gene normalized to of byperformed Reagent (Life Technologies). TRIzol using hepatocytes the SV RNA Total Isolation System (Promega). ofPreparation total ERK1/2(T202/Y204), H3(S10) Millipore;H3(S10) from anti ERK, GAPDH, anti antibodiesPrimary were: used using the “Clarity Western ECL Substrate” (BIO Substrate” ECL “Clarity the Western using withimmunoblotted the antibodies. indicated The immunoreactive were bands revealed Lysates Assay). were(BioRad SDS Protein subjected to centrifugation atmin 13000 at rpm 4°C. Proteins usingwere the Bradford quantified method andorthovanadate 30mM pyrophosphate). Supernatants sodium were collected after a 15 and inhibitor inhibitors tablet (Roche) phosphatases (5mM 2mMplus protease NaF, sodium in abuffer containing Tris anti 95 For For Total RNA were extracted RNAfrom Total were extracted For wholeFor ) , f each each f target gene was determined samples usingfrom replicate the2 formula - GSK3β, GSK3β, anti human - cyclin anti A, real - p - - Rb(S807/811 cell protein extracts,cultured cells or mouse livers were solubilized at 4°C - time PCR ona LightCycler 480 instrument (Roche) using SYBR Green anti RNA and RNA liver liver anti This article isprotected by copyright. All rights reserved. - Comment citer cedocument: p70 tissues, atissues, preliminaryRNA extraction step was using performed - - HC p - - - - Grb14 Grb14 S6K, cyclin Cruz E wereSanta Biotechnology from

GSK3β (S9), (S9), GSK3β gene expression l (50mM, pH 7,5), 150mM NaCl, 5 ) , anti anti (6) - . - 4EBP1, anti 4EBP1,

PCNA, anti PCNA, Hepatology Hepatology Quantitative measurements of transcripts were a nti mouse anti

- analysis p - - RAD) and - p 4EBP1(S65), anti - - p70 - Rb Akt, anti whole liver or from primary cultured

- from Cell Signaling anti Technology; anscriptase ( S6K(T389), anti

the - cyclin D1, a D1, cyclin

BioRad ChemiDoc MP device. - PAGE electrophoresisPAGE and mM EDTA, 1% Triton X Triton 1% EDTA, mM - time PCR (qRT time Life Technologies) - One p - - Akt(S473), anti Akt(S473), p nti -

Rb(S780) g a ue for used was μg - cyclin B cyclin ; anti The relative relative The - PCR) was PCR) - p , - Histone Histone 1 anti , anti , -

∆∆Ct - 100 and - - p p .

- - - -

GEO GSE19004 GSE19004 GEO Gene expression profiles for liver from C57Bl/6 mice injected with expression injected profiles liver C57Bl/6 mice Gene recombinant from for Results are asare Results reported means +/

L - out using Mann iver tissue fragment tissue iver BrdU antibody (1/50, ) . Samples RMA were the (Bioconductor normalized using algorithm es preparation anddata analysis - mouse biotinylatedmouse and elite antibody Vectastain (1/500) the ABC (Vector kit in situ in

were counterstained with hematoxylin. To measure proliferation, we counterstainedhematoxylin. with proliferation, were To measure

This article isprotected by copyright. All rights reserved. (8) Comment citer cedocument: cell death detection kit (Roche) following manufacturer’s instructions. manufacturer’s (Roche)following kit detection celldeath p - Whitney test or test o Whitney < )

0.05. 0.05.

fixed in 4% neut

μm thick sections. Immunohistochemistry was performed with performed was Immunohistochemistry sections. thick μm GSEA) GSEA) - DakoCytomation positive nuclei among 600 nuclei. experimentTUNEL was

(7) Hepatology Hepatology

3 - were performed

) or SE ne

ral buffered overnight wasral formalin

- M. way Differences were ANOVA. considered Grb14 (Grb14i, n= (Grb14i, Grb14 ) and)

The comparison of different was The comparison groups revelation was performed using a with with ated t these these 3 - test (limma R test (limma package). ) shRNA) were analyzed data andwith BioCarta

embedded TKO TKO affy

Page 44of54

Version postprint Page 45of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is 4. 3. 2. 1. REFERENCES 5. 7. 6. 8.

1993;262:1557 promoted by different complexproduct of E2FScience formation family members. Krek Livingston DM, Shirodkar S.W, Binding DNAto and the retinoblastoma gene 19101. a pivotalpolyploidization: role hepatocytes.for binuclear J Biol Chem 2003 JE,Guidotti Bregerie Robert A,O, Debey Brechot P, Desdouets C, Liver C. cell insulin receptor. EMBOinsulin receptor. Rep 2006;7:512 a et S, Blivet 2010;139:1355 Gastroenterology hepatocytes. Production ofal. infectious hepatitis C virus in primary of cultures adult human Podevin A, Carpentier P, Pene V, Aoudjehane L, Carrier Interaction with Grb14 Interaction V, C,N, Boute RoixZilberfarb Nouaille D, Burnol al. Blanquart Perdereau S, et J, AF, cells. carcinoma in J hepatocellular 2012;57:108 Hepatol genome etA, al. set enrichmentGene analysis: a knowledge A, Subramanian MoothaTamayo P, MukherjeeS, VK, EbertBL, MA,Gillette Paulovich Biol. Chem.J. 1998;273:26026 F, J, etGirard of al. the adapter rat Identification an as Grb14 of inhibitor insulin actions. Kasus ExpMed 201 inhibitssignaling hepatocellular carcinoma inactivation offollowing the RB pathway. J Ehmer P, Saddic U, Viatour Dorrell Andersen LA, C, JB, Zmoos Lin al. C, Notch et AF, - Van Eggelpoel MJ,Van Eggelpoel ChettouhH, Fartoux L, L, Barbu Aoudjehane V, Rey Priam C, - l. Epidermal growth factor receptor andHEREpidermall. growth receptor factor

Jacobi A,Jacobi Perdereau Auzan D, Clauser C, E, VanE, Obberghen Mauvais - wide

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- Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is BioCarta andKEGGBioCarta gene sets. syndrome and alcohol and syndrome AFP: AFP: nodules,Satellite n (%) Microvascular invasion, n (%) (%) n expression, CK19 grade Tumour n (%)Multiplicity, ( AFP Maximal fibrosis/cirrhosis,Advanced n (%) liverEtiology of chronic disease, n (%) Sex ratio (M/F) diagnosisAge at (years) Supporting from Supporting Table S1 SUPP missing four * HBV, Moderately differentiated, n (%) Well differentiated, n (%) hepatitis Combined viral NASH Hemochromatosis abuseAlcohol HBV infection HCV infection Poorly differentiated,Poorly n (%) Undetermined SD) (± Mean ≥ 400 ng/ml), n (%) a from C57Bl/6 mice injected with C57Bl/6 injected from mice hepatitis B virus; ORTING - fetoprotein; CK19: 19; cytokeratin HCV:fetoprotein;

tumour size, mean ±SD (mm)

Table Table

data

DATA

Clinicopathological characteristics of characteristics Clinicopathological *

Comment citer cedocument: ,

metabolic

See excelfile.

USi Hepatology Hepatology

( n=3) or Grb14 or n=3)

62.7 (± 12.3) 5.1 (71/14) 65.2± 38.5 13 11 (1 27 (31.8 21 (24.7 26 (30.5 41 (48.2) 21 (24.7) 18 (21.2) 15 (17.6 49 hepatitis Cvirus; hepatitis 40 (47) 17 (20) 23 8 ( 2 (2 2 5 (

( ( 15.3 57.6 9.4 5.9 (27)

2.9 .3

(GSEA) transcriptomic analysis of data ) ) )

) ) ) ) ) ) )

( n=3) n=3)

patients with HCC with patients

( GEO GSE81387

) andwith) Page 46of54 Version postprint Page 47of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is AFP: Microvascular CK19 expression Differentiation Satellite nodules Multiplicity Tumour size AFP** Advanced fibrosis/cirrhosis Alcohol +alcoholMS NASH HCV HBV characteristics of 85HCCpathological S3 Table Supporting HBV, HBV, All statistical analyses wereAll performed statistical using a Mann ** missingthree data ** missingfour data are*Values as expressed median [range] well/moderate no yes no yes 5cm ≥ cm 5 < ≥ 400 ng/ml < 400ng/ml no yes no yes no yes no yes no yes no yes no yes 5% ≥ < 5% poor

a hepatitis B virus;

fetoprotein; CK19: 19; cytokeratin HCV:fetoprotein;

invasion

This article isprotected by copyright. All rights reserved. MS, syndrome;metabolic NASH, non alcoholic steatohepatitis Comment citer cedocument: Relations between GRB14 mRNA inductionsfold (T/NT) and the

44 41 17 65 24 61 59 26 67 18 41 44 34 47 35 50 80 77 74 11 64 21 58 27 n 5 8

GRB14

0.65 0.70 0.60 [0.02 0.70 [0.0 0.67 [0.02 0.69 0.68 [0.02 0.77 0.70 [0.02 0.73 [0.02 0.47 [0.10 0.66 [0.02 0.80 0.63 [0.02 0.52 [0.02 0.70 0.53 [0.02 0.78 [0.05 0.66 0.68 [0.02 0.68 [0.02 0.80 0.79 [0.02 0.67 induction (T/NT) 0.41 0.74 Hepatology Hepatology

[0. [0.0 [0. [0. [0.05 [0. [0. [0. [0. [0.18 [0.

mRNA fold mRNA 08 05 23 29 05 08 05 05

2 5 hepatitis Cvirus; hepatitis

– – – – – – – – – – – – – – – – – – – – – – – – – –

6.50 6.50 4.39 2.31 4.39 9.42 9.42 9.42 9.42 9.42 9.42 9.42 9.42 9.42 9.42 9.42 1.68 4.3 6.50 9.42 6.50 1.06 1.6 6.50 9.42 6.50 - 9] 0] Whitney test. Whitney ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ]

P values 0. 0. 1.000 0. 0. 0.436 0.336 0. 0. 0.0 0. 0. 0.452 681 488 503 417 358 267 347 547

Results are the are Results the SEMmean 4± 3to *p<0.05. per = group). (n

were .

3 Figure Figure Figure ( - D 6 per group). ***p<0.005 for Grb14icom ***p<0.0056per group). for

on Thr389 on Thr389

) TUNEL staining of liver sections (x20). ( (x20). sections liverof ) staining TUNEL studied after 1 p<0.0 representative samples arerepresentative shown samples and GAPDH measured by blot Western

USi or Grb14i liv Grb14i USi or Results are the SEmean ± S S S littermate S4 1 2 5 compared toGrb14i for USi mice. 3

(A) (A) ( GRB14 (B) (B) A

and 4EBP1 )

qR Western blot analysisWestern Bl/6J miceBl/6J injected unspecificwith (USi) or sh Grb14 Semi

mice, mice, treated with USi or Grb14i. Results are the mean ± SEM 0.005 for Grb14i compared to USito mice compared 0.005 Grb14i for RT to to T (horizontal line), interquartile range and min to max values of

- mRNA HCC levels human andadjacent in normal livers in PCR analysis of IR of PCR analysis - 7 PCR usingpair analysiswasaunique performed primer - er sections (x20). ( (x20). sections er in liver f liver in

quantitative evaluationquantitative of IR isoforms expression in livers days

on ( Hepatology Hepatology B Ser65 rom

- is shown is C ), ), M (n = = M (n WT or after 2 after or . Q uantification of of uantification

and scanning density and Roessler liver 2 collections and . -

A andA IR 14 C Results areResults the mean ± SEM (n = 7 ) Western b Western ) pared to USi For mice. blot Western - 15 S6K DKO with mice treated USi or

E days ( ) Plasma) concentrations of alanine

per group). ***p<0.005 for Grb14i Grb14i ***p<0.005 for per group). - B expression in liver USifrom i A, A, s useds loading as control. ( B the ratio p ratio the

) Quantification of BrdU) Quantification D lot analysis of , E

; ( ). ). B ###

) (A) (A) quantification quantification

e2f1 p<0.005 for E2F1 for p<0.005 - S6K/S6K S6K/S6K Phosphorylation

expression in - liver liver adenovirus adenovirus ). Box). and and G of Rb Rb of rb14

15 15 p - Page 48of54

Version postprint Page 49of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is expression were by quantified qRT (NT).parenchyma Supporting

Figure S 5 (A) IR (A)

Relative Relative IR - A/IR and -

B ratio.B IR nontumoral adjacent liver - A and IR - B Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is This article isprotected by copyright. All rights reserved. Comment citer cedocument: Hepatology Hepatology Page 50of54 Version postprint Page 51of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is This article isprotected by copyright. All rights reserved. Comment citer cedocument: Hepatology Hepatology Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is This article isprotected by copyright. All rights reserved. Comment citer cedocument: Hepatology Hepatology Page 52of54 Page 53of54 Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is This article isprotected by copyright. All rights reserved. Comment citer cedocument: Hepatology Hepatology Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is This article isprotected by copyright. All rights reserved. Comment citer cedocument: Hepatology Hepatology Page 54of Version postprint Page 55of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo KEGG_NUCLEOTIDE_EXCISION_REPAIR KEGG_HISTIDINE_METABOLISM KEGG_HEMATOPOIETIC_CELL_LINEAGE KEGG_REGULATION_OF_AUTOPHAGY KEGG_CELL_ADHESION_MOLECULES_CAMS BIOCARTA_IL1R_PATHWAY BIOCARTA_ATM_PATHWAY KEGG_PROGESTERONE_MEDIATED_OOCYTE_MATURATION BIOCARTA_LAIR_PATHWAY KEGG_ARGININE_AND_PROLINE_METABOLISM KEGG_MISMATCH_REPAIR BIOCARTA_CELLCYCLE_PATHWAY BIOCARTA_ATRBRCA_PATHWAY KEGG_HOMOLOGOUS_RECOMBINATION KEGG_OOCYTE_MEIOSIS KEGG_VALINE_LEUCINE_AND_ISOLEUCINE_DEGRADATION BIOCARTA_G2_PATHWAY BIOCARTA_G1_PATHWAY KEGG_ABC_TRANSPORTERS BIOCARTA_MCM_PATHWAY KEGG_DNA_REPLICATION KEGG_CELL_CYCLE S1 Table Supporting BIOCARTA_STRESS_PATHWAY BIOCARTA_MYOSIN_PATHWAY KEGG_PYRUVATE_METABOLISM KEGG_GRAFT_VERSUS_HOST_DISEASE BIOCARTA_NFKB_PATHWAY KEGG_PRIMARY_IMMUNODEFICIENCY KEGG_LYSOSOME KEGG_COMPLEMENT_AND_COAGULATION_CASCADES KEGG_RETINOL_METABOLISM KEGG_SPHINGOLIPID_METABOLISM KEGG_PEROXISOME KEGG_STEROID_BIOSYNTHESIS KEGG_GLYCINE_SERINE_AND_THREONINE_METABOLISM KEGG_FATTY_ACID_METABOLISM KEGG_CYTOSOLIC_DNA_SENSING_PATHWAY KEGG_BUTANOATE_METABOLISM KEGG_DRUG_METABOLISM_CYTOCHROME_P450 KEGG_GLYCOLYSIS_GLUCONEOGENESIS KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY BIOCARTA_CSK_PATHWAY KEGG_ALANINE_ASPARTATE_AND_GLUTAMATE_METABOLISM KEGG_BASE_EXCISION_REPAIR KEGG_ACUTE_MYELOID_LEUKEMIA KEGG_PROXIMAL_TUBULE_BICARBONATE_RECLAMATION BIOCARTA_NO2IL12_PATHWAY KEGG_PYRIMIDINE_METABOLISM KEGG_PROPANOATE_METABOLISM BIOCARTA_IL7_PATHWAY KEGG_RIBOSOME KEGG_P53_SIGNALING_PATHWAY BIOCARTA_IL2RB_PATHWAY BIOCARTA_IL12_PATHWAY Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

Comment citer cedocument:

Hepatology Hepatology

- 1.9583769 1.9619515 - - 2.000575 - - - - - 2.065565 2.072623 - - 2.1064172 2.1548905 2.1728742 2.2068677 2.3205314 - 2.3639038 2.3682327 - 2.419913 2.6284354 2.7393727 NES ------1.9035201 - - - 1.9217632 - 1.94361 1.9873655 1.9953915 2.0008242 2.0053685 2.016423 2.026559 2.0459363 2.090894 2.106199 2.3625674 2.3927069 1.7413101 1.763619 1.7733407 1.7772573 1.7800556 1.7996931 1.8150942 1.8191103 1.8262639 1.8310623 1.8362898 1.844601 1.8540395 1.8662852 1.8728565 1.8816702 1.8914686 1.89343 1.8935542 1.8977716 1.9003375 1.9074398 1.9084638 1.9170498 1.9274518

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- 05

0 0 0 0 0 0 0 0 0 0

Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo KEGG_PROTEIN_EXPORT BIOCARTA_KERATINOCYTE_PATHWAY BIOCARTA_CTLA4_PATHWAY KEGG_BASAL_CELL_CARCINOMA KEGG_BETA_ALANINE_METABOLISM KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_GANGLIO_SERIES KEGG_GALACTOSE_METABOLISM KEGG_PORPHYRIN_AND_CHLOROPHYLL_METABOLISM BIOCARTA_RELA_PATHWAY KEGG_GLYCEROPHOSPHOLIPID_METABOLISM BIOCARTA_NTHI_PATHWAY BIOCARTA_PAR1_PATHWAY BIOCARTA_TOB1_PATHWAY KEGG_LEISHMANIA_INFECTION KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY KEGG_PPAR_SIGNALING_PATHWAY KEGG_SPLICEOSOME KEGG_TRYPTOPHAN_METABOLISM BIOCARTA_RACCYCD_PATHWAY KEGG_PRIMARY_BILE_ACID_BIOSYNTHESIS KEGG_TYROSINE_METABOLISM BIOCARTA_VEGF_PATHWAY BIOCARTA_CXCR4_PATHWAY BIOCARTA_NKT_PATHWAY BIOCARTA_GH_PATHWAY KEGG_VASCULAR_SMOOTH_MUSCLE_CONTRACTION KEGG_AXON_GUIDANCE KEGG_AMINO_SUGAR_AND_NUCLEOTIDE_SUGAR_METABOLISM KEGG_NOTCH_SIGNALING_PATHWAY BIOCARTA_P53HYPOXIA_PATHWAY BIOCARTA_HER2_PATHWAY BIOCARTA_AMI_PATHWAY KEGG_BASAL_TRANSCRIPTION_FACTORS BIOCARTA_INTRINSIC_PATHWAY BIOCARTA_NKCELLS_PATHWAY KEGG_INOSITOL_PHOSPHATE_METABOLISM KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS BIOCARTA_IL10_PATHWAY BIOCARTA_BARRESTIN_SRC_PATHWAY BIOCARTA_ARAP_PATHWAY KEGG_FC_EPSILON_RI_SIGNALING_PATHWAY KEGG_OTHER_GLYCAN_DEGRADATION KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION BIOCARTA_IL2_PATHWAY KEGG_B_CELL_RECEPTOR_SIGNALING_PATHWAY KEGG_VEGF_SIGNALING_PATHWAY KEGG_ETHER_LIPID_METABOLISM KEGG_CHEMOKINE_SIGNALING_PATHWAY KEGG_PRION_DISEASES KEGG_ONE_CARBON_POOL_BY_FOLATE KEGG_RNA_DEGRADATION KEGG_RIG_I_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_TYPE_I_DIABETES_MELLITUS KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_PANTOTHENATE_AND_COA_BIOSYNTHESIS KEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSIS KEGG_PHENYLALANINE_METABOLISM KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

Comment citer cedocument:

Hepatology Hepatology

- - - - - 1.6246659 ------1.6967795 - 1.7253594 ------1.4843675 - - 1.5008394 - - - 1.5255914 ------1.596521 1.6049858 - - 1.6109443 1.6126622 1.6128892 1.6138921 1.6164666 1.6256653 1.6294479 1.6354305 1.6404983 1.6421237 1.648528 1.6629981 1.6638646 1.6689674 1.6712672 1.6735142 1.6833404 1.6898562 1.6909338 1.6917739 1.723906 1.731619 1.4482245 1.4490018 1.4657335 1.4677169 1.4688942 1.469734 1.4778264 1.4807603 1.483031 1.4918332 1.4930143 1.5100654 1.5109079 1.5192238 1.532537 1.5346389 1.5402513 1.5432744 1.5447026 1.5469459 1.5527041 1.5637182 1.571226 1.5730866 1.574331 1.5852203 1.6067343 1.6074289

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Version postprint Page 57of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo KEGG_FOCAL_ADHESION BIOCARTA_TCR_PATHWAY KEGG_SMALL_CELL_LUNG_CANCER BIOCARTA_RAC1_PATHWAY BIOCARTA_BIOPEPTIDES_PATHWAY KEGG_JAK_STAT_SIGNALING_PATHWAY BIOCARTA_EDG1_PATHWAY KEGG_EPITHELIAL_CELL_SIGNALING_IN_HELICOBACTER_PYLORI_INFECTION BIOCARTA_GCR_PATHWAY BIOCARTA_SPPA_PATHWAY BIOCARTA_TOLL_PATHWAY KEGG_RNA_POLYMERASE BIOCARTA_HCMV_PATHWAY KEGG_MATURITY_ONSET_DIABETES_OF_THE_YOUNG KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY BIOCARTA_ERK_PATHWAY KEGG_ADIPOCYTOKINE_SIGNALING_PATHWAY KEGG_PURINE_METABOLISM KEGG_REGULATION_OF_ACTIN_CYTOSKELETON KEGG_LINOLEIC_ACID_METABOLISM BIOCARTA_PTDINS_PATHWAY KEGG_BIOSYNTHESIS_OF_UNSATURATED_FATTY_ACIDS BIOCARTA_TNFR2_PATHWAY KEGG_MAPK_SIGNALING_PATHWAY KEGG_ECM_RECEPTOR_INTERACTION BIOCARTA_ECM_PATHWAY KEGG_DORSO_VENTRAL_AXIS_FORMATION KEGG_PROSTATE_CANCER KEGG_MELANOGENESIS KEGG_TYPE_II_DIABETES_MELLITUS BIOCARTA_MAL_PATHWAY KEGG_GLYCOSAMINOGLYCAN_DEGRADATION BIOCARTA_MAPK_PATHWAY BIOCARTA_AKT_PATHWAY KEGG_STEROID_HORMONE_BIOSYNTHESIS KEGG_GLYCEROLIPID_METABOLISM KEGG_HEDGEHOG_SIGNALING_PATHWAY KEGG_INTESTINAL_IMMUNE_NETWORK_FOR_IGA_PRODUCTION BIOCARTA_MPR_PATHWAY BIOCARTA_BCELLSURVIVAL_PATHWAY BIOCARTA_CHREBP2_PATHWAY KEGG_CYSTEINE_AND_METHIONINE_METABOLISM BIOCARTA_41BB_PATHWAY BIOCARTA_COMP_PATHWAY BIOCARTA_TH1TH2_PATHWAY BIOCARTA_DC_PATHWAY BIOCARTA_CDC42RAC_PATHWAY KEGG_FRUCTOSE_AND_MANNOSE_METABOLISM KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION KEGG_PHOSPHATIDYLINOSITOL_SIGNALING_SYSTEM BIOCARTA_RHO_PATHWAY BIOCARTA_IL22BP_PATHWAY KEGG_GLYOXYLATE_AND_DICARBOXYLATE_METABOLISM KEGG_RENIN_ANGIOTENSIN_SYSTEM BIOCARTA_PROTEASOME_PATHWAY KEGG_ALPHA_LINOLENIC_ACID_METABOLISM KEGG_ENDOCYTOSIS KEGG_NICOTINATE_AND_NICOTINAMIDE_METABOLISM Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

Hepatology Hepatology

- 1.3412738 - - - - - 1.3685776 ------1.4082451 - - - - - 1.4304254 ------1.1912282 1.1931632 ------1.2353227 1.2359923 1.2387642 ------1.3251972 1.3389443 1.3421913 1.3433124 1.3441538 1.3505442 1.3620917 1.3753619 1.3776872 1.379137 1.3855525 1.3890727 1.3974007 1.4053646 1.407376 1.408836 1.4116176 1.415173 1.420664 1.4240769 1.4363984 1.4386209 1.169315 1.1762444 1.1814625 1.1829395 1.1857022 1.1881567 1.1994777 1.2099885 1.2106788 1.211505 1.2211521 1.2265527 1.2266266 1.2275304 1.2310311 1.2320892 1.2396324 1.240763 1.2411337 1.2617517 1.2646002 1.276081 1.29017 1.306585 1.3198428 1.3210452 1.3244259

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Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo BIOCARTA_TPO_PATHWAY KEGG_GAP_JUNCTION KEGG_AMYOTROPHIC_LATERAL_SCLEROSIS_ALS BIOCARTA_ETS_PATHWAY KEGG_PANCREATIC_CANCER BIOCARTA_CCR3_PATHWAY KEGG_METABOLISM_OF_XENOBIOTICS_BY_CYTOCHROME_P450 BIOCARTA_IL6_PATHWAY BIOCARTA_CARDIACEGF_PATHWAY BIOCARTA_MET_PATHWAY BIOCARTA_STATHMIN_PATHWAY BIOCARTA_ARF_PATHWAY KEGG_SELENOAMINO_ACID_METABOLISM BIOCARTA_NO1_PATHWAY BIOCARTA_TEL_PATHWAY KEGG_UBIQUITIN_MEDIATED_PROTEOLYSIS BIOCARTA_SHH_PATHWAY KEGG_ALLOGRAFT_REJECTION KEGG_AUTOIMMUNE_THYROID_DISEASE KEGG_MELANOMA KEGG_ENDOMETRIAL_CANCER KEGG_VASOPRESSIN_REGULATED_WATER_REABSORPTION KEGG_N_GLYCAN_BIOSYNTHESIS KEGG_HUNTINGTONS_DISEASE BIOCARTA_SPRY_PATHWAY KEGG_ERBB_SIGNALING_PATHWAY KEGG_PENTOSE_PHOSPHATE_PATHWAY KEGG_DRUG_METABOLISM_OTHER_ENZYMES KEGG_ARACHIDONIC_ACID_METABOLISM KEGG_LONG_TERM_DEPRESSION KEGG_NON_SMALL_CELL_LUNG_CANCER KEGG_THYROID_CANCER KEGG_TASTE_TRANSDUCTION BIOCARTA_IGF1MTOR_PATHWAY BIOCARTA_IGF1_PATHWAY BIOCARTA_ALK_PATHWAY KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION KEGG_STARCH_AND_SUCROSE_METABOLISM KEGG_GLYCOSYLPHOSPHATIDYLINOSITOL_GPI_ANCHOR_BIOSYNTHESIS BIOCARTA_CYTOKINE_PATHWAY BIOCARTA_INFLAM_PATHWAY BIOCARTA_FCER1_PATHWAY KEGG_APOPTOSIS BIOCARTA_MITOCHONDRIA_PATHWAY KEGG_COLORECTAL_CANCER KEGG_TGF_BETA_SIGNALING_PATHWAY BIOCARTA_FMLP_PATHWAY KEGG_CALCIUM_SIGNALING_PATHWAY BIOCARTA_HIF_PATHWAY BIOCARTA_PGC1A_PATHWAY BIOCARTA_FAS_PATHWAY KEGG_INSULIN_SIGNALING_PATHWAY BIOCARTA_ACH_PATHWAY KEGG_RIBOFLAVIN_METABOLISM BIOCARTA_PDGF_PATHWAY KEGG_GLUTATHIONE_METABOLISM KEGG_CARDIAC_MUSCLE_CONTRACTION BIOCARTA_CD40_PATHWAY Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

Hepatology Hepatology

- - 1.0768974 1.0818914 - - 1.0870419 - 1.0946121 1.0973718 ------1.1177137 - - 1.1290592 1.1309364 ------0.9300227 0.9328009 - - - 0.94418705 - - - - 0.97497576 0.9769246 ------0.9914221 - - 1.0201031 1.0228635 - - - - 1.0369705 1.0476384 - - 1.0699977 1.0708735 1.0824887 1.0865775 1.0888096 1.0991234 1.1024216 1.110977 1.1112835 1.1127336 1.1164043 1.1190732 1.1260861 1.1427759 1.1434119 1.1465533 1.1611506 0.92507607 0.92720383 0.93293 0.9383427 0.9429845 0.94568557 0.9511978 0.96681845 0.97407395 0.9794809 0.9828504 0.98718536 0.9893538 0.9895248 0.99113804 1.0021625 1.0178387 1.0246607 1.0247566 1.0253541 1.0328934 1.0487655 1.0545992

0.50031734 0.501855 0.7367232 0.7402093 0.4810396 0.47637257 0.74359846 0.4747365 0.7404559 0.75117177 0.4573714 0.45397952 0.44000086 0.4423766 0.44286773 0.4392375 0.7111043 0.4374933 0.42753482 0.69743776 0.7112896 0.39917487 0.40083832 0.398126 0.3744904 0.741166 0.74057114 0.9639633 0.9752295 0.73111326 0.7230868 0.716295 0.963425 0.71419364 0.7047854 0.6721487 0.65870047 0.8963154 0.90934175 0.6498568 0.6461438 0.6400621 0.6390864 0.6426254 0.6427485 0.8868759 0.6200977 0.58767456 0.8234814 0.8335131 0.576707 0.58013254 0.58227557 0.56936646 0.8119731 0.7999242 0.53855234 0.529016 Page 58of54

Version postprint Page 59of54 associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo BIOCARTA_BAD_PATHWAY BIOCARTA_MTOR_PATHWAY KEGG_RENAL_CELL_CARCINOMA KEGG_GNRH_SIGNALING_PATHWAY BIOCARTA_CALCINEURIN_PATHWAY BIOCARTA_CK1_PATHWAY BIOCARTA_EIF4_PATHWAY KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_CHONDROITIN_SULFATE KEGG_GLIOMA KEGG_WNT_SIGNALING_PATHWAY KEGG_PATHWAYS_IN_CANCER KEGG_BLADDER_CANCER BIOCARTA_EGF_PATHWAY KEGG_ADHERENS_JUNCTION BIOCARTA_PPARA_PATHWAY BIOCARTA_CDMAC_PATHWAY KEGG_LYSINE_DEGRADATION BIOCARTA_INSULIN_PATHWAY BIOCARTA_GLEEVEC_PATHWAY BIOCARTA_TID_PATHWAY BIOCARTA_NGF_PATHWAY KEGG_HYPERTROPHIC_CARDIOMYOPATHY_HCM BIOCARTA_UCALPAIN_PATHWAY BIOCARTA_GATA3_PATHWAY KEGG_NEUROTROPHIN_SIGNALING_PATHWAY BIOCARTA_MCALPAIN_PATHWAY KEGG_PARKINSONS_DISEASE KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_LACTO_AND_NEOLACTO_SERIES BIOCARTA_HIVNEF_PATHWAY BIOCARTA_TFF_PATHWAY BIOCARTA_DEATH_PATHWAY BIOCARTA_WNT_PATHWAY BIOCARTA_PYK2_PATHWAY KEGG_ARRHYTHMOGENIC_RIGHT_VENTRICULAR_CARDIOMYOPATHY_ARVC KEGG_OLFACTORY_TRANSDUCTION BIOCARTA_IL3_PATHWAY BIOCARTA_GPCR_PATHWAY BIOCARTA_RAS_PATHWAY KEGG_CITRATE_CYCLE_TCA_CYCLE KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_HEPARAN_SULFATE KEGG_ALZHEIMERS_DISEASE KEGG_OXIDATIVE_PHOSPHORYLATION BIOCARTA_VIP_PATHWAY BIOCARTA_AGR_PATHWAY BIOCARTA_P38MAPK_PATHWAY KEGG_O_GLYCAN_BIOSYNTHESIS KEGG_ALDOSTERONE_REGULATED_SODIUM_REABSORPTION KEGG_PROTEASOME BIOCARTA_NFAT_PATHWAY KEGG_VIRAL_MYOCARDITIS BIOCARTA_IGF1R_PATHWAY KEGG_MTOR_SIGNALING_PATHWAY BIOCARTA_AT1R_PATHWAY BIOCARTA_CASPASE_PATHWAY BIOCARTA_BCR_PATHWAY KEGG_PATHOGENIC_ESCHERICHIA_COLI_INFECTION KEGG_ASTHMA BIOCARTA_CTCF_PATHWAY Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

Hepatology Hepatology

- - - - - 0.8032015 0.8077915 - - 0.8281449 - - - 0.860394 - 0.862868 0.86902344 - - 0.87663275 ------0.6552235 0.65528196 0.65777916 ------0.6848396 - - 0.6930238 - - - - 0.7222216 - 0.7228392 - - - 0.74268 0.7505752 0.7511516 0.752076 0.75624263 0.76383954 0.7758864 - 0.77817005 0.7801709 0.78565675 0.78573745 0.7870396 0.8086713 0.82002115 0.8404916 0.8474534 0.85661364 0.8615491 0.86952966 0.87148243 0.8983203 0.898462 0.9021251 0.9202868 0.9214274 0.65180737 0.6603868 0.6623327 0.6689508 0.66950035 0.67072845 0.6764886 0.677364 0.6848684 0.69172525 0.6944882 0.7007401 0.71357554 0.71837944 0.7222655 0.7239398 0.7263676 0.7351197 0.7773184

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Version postprint associated with hepatocellular carcinoma. Hepatology, 65 (4), 1352-1368. DOI :10.1002/hep.28972/suppinfo BIOCARTA_CHEMICAL_PATHWAY BIOCARTA_NOS1_PATHWAY KEGG_VIBRIO_CHOLERAE_INFECTION BIOCARTA_HDAC_PATHWAY KEGG_LONG_TERM_POTENTIATION BIOCARTA_MEF2D_PATHWAY BIOCARTA_GSK3_PATHWAY BIOCARTA_CARM_ER_PATHWAY BIOCARTA_CREB_PATHWAY KEGG_TIGHT_JUNCTION BIOCARTA_ERK5_PATHWAY BIOCARTA_EPO_PATHWAY KEGG_SNARE_INTERACTIONS_IN_VESICULAR_TRANSPORT BIOCARTA_PML_PATHWAY BIOCARTA_TNFR1_PATHWAY KEGG_DILATED_CARDIOMYOPATHY KEGG_AMINOACYL_TRNA_BIOSYNTHESIS BIOCARTA_INTEGRIN_PATHWAY BIOCARTA_PTEN_PATHWAY KEGG_CHRONIC_MYELOID_LEUKEMIA BIOCARTA_TGFB_PATHWAY BIOCARTA_CCR5_PATHWAY BIOCARTA_CERAMIDE_PATHWAY BIOCARTA_ACTINY_PATHWAY BIOCARTA_NDKDYNAMIN_PATHWAY BIOCARTA_RARRXR_PATHWAY Morzyglod, L.,Caüzac, M.,Popineau, L.,Denechaud, P.-D.,Fajas, L.,Ragazzon, B.,Fauveau, V., Planchais, J.,Vasseur Cognet,M., Fartoux,L.,Scatton, O.,Rosmorduc, O.,Guilmeau, S., Postic, C.,Desdouets, C., Desbois Mouthon, C., Burnol, A.-F.(2017). Growth factorreceptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is

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Hepatology Hepatology

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