Mitochondria uia Ca luminal fioio ,,-rshsht (IP 1,4,5-trisphosphate inositol of oto oeoelpigfntoswt a,adatrdCa altered and Ras, with functions overlapping some control ppoi a nacda euto K-Ras of result a as enhanced was apoptosis noei -a upessIP suppresses K-Ras Oncogenic ARTICLE RESEARCH eevd9Spebr21;Acpe 2Jnay2014 January 22 Accepted 2013; September 9 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This Open ` UK. The 6AA, Sciences, MK7 Chemical and Keynes UK. Health Milton 3AT Life, University, of CB22 Department Cambridge address: Campus, upstream *Present Research Babraham as Institute, significant position Babraham their elicit to owing mutations fate (RTKs) These cell kinases for 2003b). tyrosine consequences (Downward, ( receptor itself in Raf Ras in mutation EGFR), de-regulated to (e.g. frequently 2003b; is due pathway Downward, This cancer 2004). (Cully 2003a; al., kinases Downward, et protein Schulze 2008; Raf the Downward, of and receptor upstream of and downstream kinases switches tyrosine molecular as serve proteins Ras INTRODUCTION IP 1,4,5-trisphosphate, Inositol Cancer, K-Ras, WORDS: KEY Ca of suppression that suggest results Ca intracellular K-Ras (ER) of loss reticulum by enhanced endoplasmic is stores the from release in players important as Ca how emerging transformation, are oncogenic pathways Ca signalling Although phenotype. cell Ras in associated underlying changes pathways. cancer, in that deregulated signalling frequently kinases is signalling cell-fate-determining of tyrosine downstream multiple receptor engaged controls switch and molecular receptors a G-protein-coupled is Ras GTPase The Roderick Llewelyn H. and ABSTRACT Bootman* D. Martin Foets, F. C. Thomas Cook, J. Simon Pierro, Cristina hog eoeln fteioomcmoiino IP of composition isoform the of remodelling through hscnrbtst uvvlavnaedrn oncogenic during advantage survival a to transformation. contributes this by deleted Ca was agonist-induced that (G13D) show K-Ras We which oncogenic recombination. in derivatives homologous encoding isogenic allele their and the lines cell cancer colorectal epnet aual curn eeso K-Ras of levels occurring naturally to response uigtasomto n hte thsacua oeremains role Ca causal investigated a have has it We whether unclear. and transformation during ossety paeo Ca of uptake Consistently, Ca uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,10–69doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2+ otn fteE tr n oiiaino h abundance the of modification a and store ER the of content 2+ , 2+ eesi ooetlcne ellines cell cancer colorectal in levels 0 falhmncnes n B- and cancers) human all of 20% nomtcodi n estvt to sensitivity and mitochondria into G13D 3 eetr(PR subtypes. (IP3R) receptor ) 2+ 2+ 2+ inligi w human two in signalling hog nices nthe in increase an through inligi remodelled is signalling inligi common a is signalling 2+ inligpathways signalling G13D G13D os These loss. n that and , 3 ,Ca 2+ 2+ 2+ 2+ , 3 dpnetCa -dependent iohnrai niiitro h nrni ppoi cascade of apoptotic deregulation intrinsic therefore, surprisingly the Not Ca 2003). of al., initiator et (Rizzuto an is mitochondria enrpre nvrostmus(otihe l,2007), al., et Ca of (Monteith principles general tumours formulate various to attempts in but reported been eudnyi ucinbtenteesga transduction targeting drug signal overcome these to cells between transformed allows function cascades 2013). in al., et Little redundancy 2012; Flaherty, and approved (Belden been clinic now the have in pharmaceutical drugs pathway and Indeed, the B-Raf–Mek–Erk therapeutics. the by cancer downstream targeting of Hanahan targeted the development been the and in 2000; have Ras industry of engages role it Weinberg, central pathways This and transformed 2011). the Weinberg, of (Hanahan hallmarks – cell death phenotype of cell regulation and the metabolism in cycle, involved pathways multiple of regulators neato nocgnctasomto sta ewe a and Ras between that signalling is undefined, Ca transformation yet as oncogenic but in major, A interaction 2013). Ras-driven al., of et suppression (Wu for cancer for these essential strategies therefore effective is of of and pathways development Ras signalling Many between cellular interactions al., major 2013). of et other (Wu nature al., cancer the in Understanding et dysregulated 2013). also (Little are pathways resistance downstream develop and enpooe ob upre yarmdligo hi Ca their of remodelling a by supported have be Weinberg, characteristics to these and proposed 2011); (Hanahan been Weinberg, and evasion uncontrolled Hanahan by apoptosis 2000; insufficient part in or and characterized increased are proliferation cells and Cancer death. proliferation cell diminished or overt leain ncne aetu a ald itecnitnyi the in consistency Ca Little failed. in far alterations thus have cancer in alterations 02 aladMas 03,weesCa whereas 2003), Means, Lockyer, and and (Cullen Kahl (CaM) calmodulin 2002; via cycle, cell the sustain oCa to consequence. a is or phenotype cancer Ca of number a of alterations expression Indeed, the 2008). Cook, in and (Roderick toolkit signalling uor n ewe tde.Mroe,fwsuishave studies few Ca Moreover, altered studies. whether between clarified and tumours hw oehneaoitrgltdioio 1,4,5-trisphosphate inositol agonist-regulated (IP enhance was Ras to example, For shown pathways. these between interplay complex ta. 96,arsl htmgtb wn oteaiiyo Ras of ability the to (PLC) owing C be phospholipase might bind that to result protein a 1986), al., et e oe nlf n et hie,icuigtedcso to Ca (Berridge decision cytoplasmic 2003) in the al., Oscillations et including 1998). (Berridge choices, al., apoptosis death et by and die or life proliferate in roles key sRsle ptemo utpeclua pathways, cellular multiple of upstream lies Ras As ohr sti oeaprn hni tde ikn Ras linking studies in than apparent more this is Nowhere Ca 2+ 2+ 3 rdcin(ahie l,19;Ln ta. 91 Wakelam 1991; al., et Lang 1993; al., et (Hashii production ) . 2+ oesai a enrpre ndsae soitdwith associated diseases in reported been has homeostasis 2+ sapeorpcsgaln esne ht ieRs plays Ras, like that, messenger signalling pleiotropic a is inlig hc obc vr2 er n eela reveal and years 25 over back go which signalling, 2+ rti xrsini on mn different among found is expression protein 2+ 2+ inligcnrbtst the to contributes signalling release 3 2+ sadER and Rs hnln rtishave proteins -handling e Bne ta. 2006; al., et (Bunney 2+ 2+ r eesr to necessary are vrodb the by overload ` 2+ signalling 1607 2+

Journal of Cell Science hssuy esuh oaayeteefcso igeoncogenic compared single we a end, this of To effects levels. the expression physiological analyse at In to allele sought 2004). we al., are study, et oncogenes this (Tuveson pleiotropic levels which supra-physiological in at with expressed associated systems issues experimental the of and great studied use cells experimental this cancer appropriate the identifying to for in controls Contributing problem 2008). the is Cook, variability and (Roderick lacking Ca upon oncogenes ‘driving’ the of expressed effect natively the of of role and presence nature the Moreover, transformation. eue RCa ER reduces otiue osm ftesrkn el n tissue-specific and cell- Ca striking of the regulation Weinberg, undoubtedly the of Ras in and some differences of (Hanahan pleiotropy to cell of This promoting cell contributes all including survival. 2011), cancer and virtually Weinberg, proliferation the to and Hanahan contribute of 2000; and hallmarks pathways the effector and activate (Bollag oncoproteins several active Ras permanently de-regulated These being GTP 1991). Ras of McCormick, hydrolysis in Ras the resulting prevent GAPs, Gly13 of which by Gly12, variants 1987), at the (Barbacid, substitutions oncogenic all Gln61 missense mutant or normal made harbouring expressing is those The as and cells such 2008). complex in or Cook, is so (Cook events promote more and these signalling Roderick between to Ras-dependent interplay 2006; and (GAPs) Lockyer, Ras proteins and of activation GTPase-activating inhibit Ras (GEFs) factors or guanine-nucleotide-exchange Ras certain activate noei transformation. oncogenic unyadKtn 06.Cnesl,Ca Conversely, 2006). Katan, and Bunney ARTICLE RESEARCH 1608 Ca regarding how consensus such, As determined. been not has homeostasis proteins of number a of expression Ca in in involved alterations Although IP RESULTS of levels occurring naturally to response K-Ras common a is signalling eie ellnshv endsrbd(otihe l,20) few Ca 2007), how al., analysed et (Monteith have described studies been have lines cell derived Ca of suppression that suggest results These mitochondria. K-Ras elln ar nwihthe which in pairs line cell avoid overexpression. to from Ras mutant results arising of confirm artefacts levels expression to native important with is systems cell it in merits, of have overexpression proteins conditional Ras mutant employing fibroblasts studies 2004). although embryo al., cell Thus, et causes (Tuveson mouse transformation levels oncogenic primary endogenous and proliferation at in expression strikingly, is different conditional senescence Most cycle whereas quite it cell and level. elicits elicit K-Ras however, expression arrest oncogenic can of their addition, overexpression proteins on conditional In Ras depending mutant mutants. effects that Ras apparent oncogenic with xrsin sn hsapoc,w hwta noei K-Ras single oncogenic that This a Ca show of Ras. we inhibits approach, effects oncogenic this the of Using mutant comparing expression. lack of of the benefit copy from the derivative apart has isogenic an approach identical with mutant is compared the directly harbouring that be line cell then cancer can (Shirasawa parental allele recombination The homologous 1993). by al., et ablated been has Ras 3 ooecm hsise ehv ae datg fisogenic of advantage taken have we issue, this overcome To idcdCa -induced G13D G13D 2+ 2+ inligpriiae nclua rnfrainis transformation cellular in participates signalling htcnrbtst uvvlavnaeduring advantage survival a to contributes that 2+ ees rmteedpamcrtclm(ER), reticulum endoplasmic the from release 2+ 2+ euaini aiu uortpsadtumour- and types tumour various in regulation KRAS ees sicesdi el eee of deleted cells in increased is release eesadsprse Ca suppresses and levels ahrta sn supra-physiological using than rather KRAS 2+ inligi lee sarsl of result a as altered is signalling leeecdn noei K- oncogenic encoding allele 2+ inligse nstudies in seen signalling 2+ 2+ inligcan signalling lxt the to flux 2+ 2+ h C16clrca acrcl ie(K-Ras line cell cancer colorectal HCT116 the hog iia GPCR–G similar a through netgtdwehrCa whether investigated 3B). (Fig. counterparts ietyeggd yasn PR G GPCR, bypassing engaged, directly ellnst vlaewehrtepeec fteendogenous the of presence the whether K-Ras evaluate oncogenic these to employed We lines 1993). al., form cell et not (Shirasawa do mice and agar nude soft in in tumours grow not do cells HKH2 to cells, contrast HCT116 In recombination. homologous by deleted been has allele hs aateeoeidctdta K-Ras that indicated therefore data These h nraei Ca in increase The Fg Ai)adi h nerl(raudrtecre U)of AUC) amplitude curve; the the under the (area in integral in 1Aii), the Ca (Fig. evident the in was cells and 1Aiii) difference responding (Fig. This of 1Ai). to percentage (Fig. compared when cells enhanced HCT116 were ATP with receptors purinergic otiue oteehneeto RCa ER of enhancement the to contributed ematetrfe omo IP of form esterified permeant a cigt upesCa suppress to acting was vrteclsi Ca in cells the over C16clswe Ca when cells HCT116 si xeiet sn T n itmn,Ca histamine, and ATP using experiments in As ecnaeo epnigclsadi h mltd n U of AUC and amplitude the in Ca and the cells responding of percentage Ca hsaoit rae ecnaeo K2clsehbtdCa exhibited cells HKH2 of percentage greater a agonist, this uiegcrcpo inligbtwsamr eea effect, general more a engagement. GPCR was of downstream but signals involving signalling receptor purinergic inl.A hw nFg ,a osqec fls fK-Ras Ca of loss cells, of consequence HKH2 a as in 1, Fig. in shown As signals. bevdi C16cls(i.3) hs aaidctdta the Ca that indicated data in These those 3A). alteration than (Fig. AUC cells and HCT116 amplitude in greater observed a of were which transients, HKH2 of result a as generation. developed initial phenotype their since a culture not and ablation significant a in resulted cells HCT116 Ca in ATP-stimulated siRNA in Ras 2A). increase (Fig. of Ras siRNA non-targeting cells depletion approach, suppressed HCT116 control to this also compared with Using when transfected 85% was (siRNA). by reduced RNA expression was expression interfering Ras small HCT116, by in signalling fK-Ras of xrclua Ca extracellular akn K-Ras lacking U n epnieest gns apida concentration observed Ca were a responses agonist at in in (applied differences greatest agonist the to where responsiveness and AUC yIP by ht eai(uyyoyehl;IP hexakis(butyryloxymethyl); phate sgncdrvtv K2(K-Ras HKH2 derivative isogenic ifrnebtenHT1 n K2clsi u oK-Ras the to due that is conclusion cells the HKH2 support and HCT116 and between cells difference HKH2 in observed that rti-ope eetr(PR inligi hs el,we cells, Ca these in histamine-induced signalling examined (GPCR) receptor protein-coupled ta. 00.Uigti prah IP Thomas approach, 2004; al., this et Using Kasri 2000). 2006; al., al., et et (Conway experiment ie htKRsi rqetymttdi ooetlcne,we cancer, colorectal in mutated frequently is K-Ras that Given odrcl drs hte natrto nIP in alteration an whether address directly To Ca enhanced the whether test To ofrhrpoeteitrcinbtenRsadCa and Ras between interaction the probe further To oioaetecnrbto fCa of contribution the isolate To 2+ 3 rnin,eprmnswr efre nteasneof absence the in performed were experiments transient, Mwr logetri K2clsta nterHCT116 their in than cells HKH2 in greater also were BM 2+ 2+ 2+ ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal cnann ufr eandgetri K2than HKH2 in greater remained buffer) -containing G13D rnins(i.1Aiv). (Fig. transients epne Fg Bii) hs aaaecnitn with consistent are data These 2Bii–iv). (Fig. responses eeini C16clswsagnrlfaueo G- of feature general a was cells HCT116 in deletion G13D 2+ 2+ G13D sosre nCa in observed As . 2+ 2+ ,Ca 2+ lxswsntseii odfeecsin differences to specific not was fluxes fe mgn ufrdrn h oreo the of course the during buffer imaging -free inligwsmnfs sa nraei the in increase an as manifest was signalling inl nue olwn tmlto of stimulation following induced signals leemdfe h eeaino Ca of generation the modified allele 2+ 2+ 2+ 2+ a mte rmteiaigbuffer. imaging the from omitted was inligwsas eoelda a as remodelled also was signalling q ees a nue ihacell- a with induced was release ees rmteE Fg 1B). (Fig. ER the from release –PLC–IP 2+ 3 2 inl nteecls(i.2B). (Fig. cells these in signals 2+ 2+ 2+ /WT moioio 1,4,5-trisphos- [myo-inositol epne bevda result a as observed responses nlxt h agonist-induced the to influx 3 q epne,wihproceed which responses, M,wihwsperfused was which BM], 3 nwihtemttdRas mutated the which in ) L n noeosIP endogenous and PLC , 2+ aha.I epneto response In pathway. 3 cnann ufr the buffer, -containing G13D eetr (IP receptors 2+ 2+ G13D/WT nHT1 cells HCT116 in ees ncells in release inl induced signals 3 signalling )withits 3 s are Rs) G13D G13D 2+ 2+ 2+ 3 .

Journal of Cell Science nbt C16adDD1cells. DLD-1 and HCT116 both in nHH el a u oa nraei otn fthe of content in increase an to due was cells HKH2 in htocgncK-Ras oncogenic that EERHARTICLE RESEARCH ie httemgiueo Ca of magnitude the that Given Ca ER K-Ras the in greater were fluxes K-Ras of loss of result Ras u nlsst Ca to analysis our eemndb t tt fflig ehpteie htthe that hypothesized we filling, of state Ca its enhanced by determined Ras ecnaeo epnigclsadi h mltd n U of AUC and amplitude the in Ca and the cells responding of percentage eecridoti Ca experiments in derivatives, out isogenic performed their carried As and 1993). were al., cells et HCT116 (Shirasawa for DLD-1 line, cell cancer G13D/WT 2 /WT 2+ 2+ hni hi aetlioei L- elln (K- line cell DLD-1 isogenic parental their in than ) otn sicesdi el eee fK-Ras of deleted cells in increased is content epne Fg i–v.Tkntgte,teedt show data these together, Taken 4ii–iv). (Fig. responses Fg ) hswsmnfs sa nraei the in increase an as manifest was This 4). (Fig. ) 2+ ees bevdfloigK-Ras following observed release 2+ G13D ees rmteE.APidcdCa ATP-induced ER. the from release G13D iisIP limits 2+ nascn needn colorectal independent second a in fe mgn ufrt restrict to buffer imaging -free G13D 2+ 3 idcdCa -induced dltdDO elln (K- line cell DKO4 -deleted eesdfo h Ris ER the from released 2+ ees (IICR) release G13D deletion G13D 2+ IP antd fteCa the of magnitude rmteE asn Ca causing ER the from ebae esrmnswr efre nCa in performed were measurements membrane, Rwsas esrddrcl sn eeial noe GFP- encoded genetically Ca a using based directly measured also was ER Ple ta. 04.Ti niao eisuo Ca a upon relies indicator This 2004). al., et (Palmer aueo sn h gidcdeeaini yooi Ca cytosolic in elevation Tg-induced the using of nature to compared 5Aii,iii). line (Fig. cell cells HKH2 HCT116 the in greater significantly however, mltd n U fteT-nue Ca Tg-induced the of AUC and amplitude upihbtrtasgri T)wsaaye.Through Ca non-specific analysed. the was reveals Tg (Tg) SERCA, thapsigargin of inhibition inhibitor pump esr fE uia Ca luminal ER of measure ufr plcto fT nue neeaini intracellular in elevation an induced Tg Ca of Application buffer. elto ihT lolast Ca to leads also Tg with depletion 3 ocmlmn hs aaadt comdt o h indirect the for accommodate to and data these complement To 2+ rlaal RCa ER -releasable nbt h C16adHH ellns(i.5) The 5A). (Fig. lines cell HKH2 and HCT116 the both in ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal 2+ niao agtdt h R(nw sD1ER) as (known ER the to targeted indicator el e oesi eeaaye.* analysed. ** were coverslip per cells yewr mgdo ahdy( day each cell on per imaged coverslips were three type where experiments, of days fetaellrCa extracellular presence of the in performed concentrations experiments the and at indicated ATP with stimulated were i.1 gns AP-nue Ca (ATP)-induced Agonist 1. Fig. Ca line. cell cancer el.Rslsaetemeans the are Results cells. el.(ii ekapiueo Ca of amplitude Peak (Aiii) cells. C16clsadbakbr ersn K2cells. Ca HKH2 ATP-stimulated represent Representative bars (Bi) black represent and bars cells White HCT116 analysed. were ( coverslip day per type each cell on per imaged coverslips were three where experiments, of means are Results cells. Av nertdCa Integrated (Aiv) Bi ecnaeo epnigcls Bi)Peak Ca (Biii) of cells. amplitude responding of Percentage (Bii) nlecdb K-Ras by influenced nHT1 n K2cls el eestimulated were Cells 2 cells. with HKH2 and HCT116 in P 2+ 2+ , 2+ .1 *** 0.01; epne fHT1 n K2cls Cells cells. HKH2 and HCT116 of responses oiie rmteE yteSERCA the by ER the from mobilized 2+ m tr.T sesE Ca ER assess To store. T nasneo xrclua Ca extracellular of absence in ATP M 2+ cuuaini h yoo.A store As cytosol. the in accumulation otn,tefe Ca free the content, P , 2+ .0 (Student’s 0.001 A)Rpeettv ATP-stimulated Representative (Ai) epne Bv U fresponding of AUC (Biv) response. 2+ 2+ 2+ Ai ecnaeo responding of Percentage (Aii) . G13D epne(U)o responding of (AUC) response nlxars h plasma the across influx 6 ...o aafo days 4 from data of s.e.m. nteHT1 colorectal HCT116 the in n 6 5 ...o aafo 3 from data of s.e.m. 2.A es 0cells 60 least At 12). 2+ n 2+ 5 2+ t -test). 2+ 2+ response. P ) tlat60 least At 9). rnin was, transient 2+ otn fthe of content fe imaging -free , 2+ inl are signals 2+ 0.05; -dependent ees the levels, responses 2+ 2+ 2+ 1609 . leak sa as

Journal of Cell Science nacdCa enhanced w eltps ai fbslFE oCa to the FRET in basal equivalently of ratio behaving A was types. D1ER cell two that indicated types plcto fteCa the of Application lta,wihwseuvln ewe h w eltypes cell two Ca the similar between The equivalent 5Bii). was (Fig. which plateau, Ca rae Ca greater Fg 5Biii). (Fig. EERHARTICLE RESEARCH 1610 Ca ER in increase an that identified Having IP line, cell ratio) HCT116 the YFP:CFP in Ca (the than basal line FRET higher cell ER indicating HKH2 greater the the cells, in observed with localization resting was ER colocalized In its was confirming and 5Bi). types, D1ER (Fig. cell pattern GFP. both in reticular of calnexin protein derivatives a (YFP) in yellow expressed and (CFP) cyan Fo in change hrfr sdt omlz RCa ER normalize to used therefore xrsini nrae nclsdltdo K-Ras of deleted cells in increased is expression 3 2+ sfr xrsini eoeldadSERCA2b and remodelled is expression isoform R rmteE tr eutdi eln nFE oalower a to FRET in decline a in resulted store ER the from 2+ 2+ rtrrsnneeeg rnfr(RT between (FRET) transfer energy resonance ¨rster eesi h Ro K2ta C16cells HCT116 than HKH2 of ER the in levels inligi K-Ras in signalling 2+ 2+ oohr ooyi oflydeplete fully to ionomycin ionophore nteE fti elln Fg 5Bii). (Fig. line cell this of ER the in 2+ fe RTbtenbt cell both between FRET -free 2+ G13D ees hc loindicated also which levels, dltdcls nanalysis an cells, -deleted 2+ 2+ otiue othe to contributed fe RTwas FRET -free G13D hra h xrsino artcln h ao Ca major the calreticulin, of expression the whereas loivsiae u on ob nhne nclslacking cells in was unchanged expression be its to 2005), found al., K-Ras but been et investigated has (Brouland two upregulation also cancer SERCA3 the that in between Given altered reported 5D). not (Fig. was types cells, cell non-excitable in protein K2adHT1 el.Seiial,IP Specifically, cells. HCT116 and HKH2 xrsinpoieo IP of profile expression fpoen novdi RCa ER in involved proteins of TR)epeso a nrae n IP and increased was expression ITPR3) xrsino EC2,wihi rmrl epnil for The responsible primarily S1). is Fig. which Ca material SERCA2b, ER – profile of (supplementary of proteins expression expression other immunoblotting Expression two similar did in types. a as cells and cell exhibit of HKH2 GAPDH and normalization to between HCT116 for found between interest control was protein (Drawnel loading of membrane calnexin mass a ER protein when the as molecular 2009), the used elsewhere high al., was et a employed Harzheim calnexin of 2012; have al., proteins et we ER as analysing experiments, these fdt rm3dy feprmns hr he oesisprcell per ( * coverslips day analysed. three each were where on experiments, imaged of were means days type are 3 Results from cells. data responding of of AUC (Biv) response. Ca ATP-stimulated 2. Fig. C16clssiuae ih2 with stimulated cells HCT116 Bi ecnaeo epnigcls Bi)Pa mltd fCa of amplitude Peak (Biii) cells. responding of Percentage (Bii) oto.(i)Qatfcto fKRskokoni C16cells HCT116 in loading knockdown a K-Ras ( as of or used Quantification siRNA was (Aii) (ctrl) (CLNX) control. control Calnexin with K-Ras. transfected targeting cells siRNA HCT116 in K-Ras of cells. HCT116 in K-Ras of depletion n G13D 2+ 5 ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal ) B)Rpeettv Ca Representative (Bi) 3). eusrto,wsicesdi K2cls(i.5C), (Fig. cells HKH2 in increased was sequestration, splmnaymtra i.S) oal,the Notably, S1). Fig. material (supplementary b atn–ta r otnl sdfrnormalization for used routinely are that – -actin P , .5 ** 0.05; 3 swssgiiatydfeetbetween different significantly was Rs 2+ P , 2+ epne r nacdb siRNA by enhanced are responses 2+ .1 *** 0.01; m n epne fsiRNA-transfected of responses T nCa in ATP M 5 inligwscridot In out. carried was signalling ) tlat6 el e coverslip per cells 60 least At 9). A)Rpeettv immunoblot Representative (Ai) P , .0 (Student’s 0.001 3 3 1(lokonas known (also R1 2+ 3(lokonas known (also R3 fe mgn buffer. imaging -free 2+ t -test). 6 storage s.e.m. 2+

Journal of Cell Science itmn a ple nteCa the in applied was Histamine nw sIP2 xrsinwsntdtcal nete cell either in S1). detectable Fig. not material was (supplementary expression type ITPR2) as known EERHARTICLE RESEARCH TR)epeso eue nteHH K-Ras HKH2 the in reduced expression ITPR1) Ca 3. Fig. el hncmae oHT1 el Fg E.IP 5E). (Fig. cells HCT116 to compared when cells xeiet,weetrecvrlp e eltp eeiae nec day each on Ca imaged were type cell ( per coverslips three where experiments, aso xeiet,weetrecvrlp e eltp eeiae on imaged were type *** cell per ( coverslips day three each where experiments, of days Ca Bi ecnaeo epnigcls Bi)Pa mltd fCa of amplitude Peak (Biii) cells. responding of Percentage (Bii) epnigcls Ai)Pa mltd fCa of amplitude Peak (Aiii) cells. responding epnigcls eut r means are Results cells. responding nacdi h K-Ras the in enhanced Bv U frsodn el.Rslsaemeans are Results cells. responding of AUC (Biv) n 5 P 2+ 2+ 2.A es 0clsprcvrlpwr nlsd B)Representative (Bi) analysed. were coverslip per cells 30 least At 12). , rcso C16adHH el xoe oIP to exposed cells HKH2 and HCT116 of traces 1 to exposed cells HKH2 and HCT116 of traces .0 (Student’s 0.001 2+ n 5 epne nue yhsaieadIP and histamine by induced responses 2.A es 0clsprcvrlpwr nlsd ** analysed. were coverslip per cells 60 least At 12). t -test). G13D dltdHH elline. cell HKH2 -deleted 2+ fe mgn ufr Ai ecnaeof Percentage (Aii) buffer. imaging -free 6 ...o aafo asof days 4 from data of s.e.m. 2+ epne Av U of AUC (Aiv) response. 6 ...o aafo 4 from data of s.e.m. m 3 A)Representative (Ai) histamine. M 3 se (10 ester se are ester G13D 2+ 3 2(also R2 response. m -deleted P M). , 0.01; noei rnfrain(aaa n eneg 2000; IP in Weinberg, enhancement the during Given and remodelled 2011). properties Weinberg, (Hanahan cell and Hanahan – transformation 2001) al., oncogenic et Pinton 2010; Ca 2006). Pozzan, and Rizzuto 1998; rm3dy feprmns hr he oesisprcl yewere type cell per ( * coverslips day three each where on experiments, imaged of days 3 from h orefrmtcodilCa mitochondrial for source the eehne n otiuet rae estvt odeath- to sensitivity greater K-Ras to the in contribute stimuli and inducing enhanced be rvlgdmne tmcooan fhg Ca high of microdomains at manner privileged a IP nueeuvln Ca equivalent induce oei euaino eldahadmtbls (Ca metabolism and death cell of regulation in role (Csorda proximity epne eedtce ymauigtersda non- Ca and cytosolic analysed bulk residual be for this can surrogate cell the a In the as nucleus. of used measuring region the mitochondrial-free a in by way, fluorescence detected rhod-2 compartmentalized were responses Ca Mitochondrial K-Ras of deleted Cells means are Results cells. responding of AUC (Aiv) response. Ca ATP-stimulated 4. Fig. aia ocnrto fAPwsapidadclswere cells and applied Ca was in ATP of imaged concentration maximal tmltdCa stimulated Ca mediated IP rmIP from Ca el,w yohszdta iohnra Ca mitochondrial that hypothesized we cells, -a eeinuo iohnra Ca mitochondrial upon deletion K-Ras oprmnaie hd2A Fg A.CtpamcCa Cytoplasmic 6A). (Fig. AM rhod-2 compartmentalized Ca ufr Ai ecnaeo epnigcls Ai)Pa mltd fCa of amplitude Peak (Aiii) cells. responding of Percentage (Aii) buffer. h L- ooetlcne elline. cell cancer colorectal DLD-1 the fDD1adDO el tmltdwt 2 with stimulated cells DKO4 and DLD-1 of P , 3 3 2+ 2+ st h iohnrahsbe hw opa nimportant an play to shown been has mitochondria the to Rs close in are mitochondria and ER the where sites at located Rs .5 *** 0.05; paeadsniiiyt apoptosis to sensitivity and uptake a esrdb ofcliaigo mitochondrially of imaging confocal by measured was ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal 3 s xeietlcniin eeue nwiha which in used were conditions experimental Rs, P , 2+ .0 (Student’s 0.001 2+ inligosre nK-Ras in observed signalling 2+ ees rmteE a hrfr nlsd To analysed. therefore was ER the from release 2+ fe mgn ufr iohnra matrix Mitochondrial buffer. imaging -free ´ ta. 06 uhn 00 izt tal., et Rizzuto 2000; Duchen, 2006; al., et s n 5 paei o-fiiypoesta cusin occurs that process low-affinity a is uptake 2+ ) tlat6 el e oesi eeanalysed. were coverslip per cells 60 least At 9). 2+ inl ewe eltps n orestrict to and types, cell between signals G13D epne r otoldb K-Ras by controlled are responses t xii nrae mitochondrial increased exhibit -test). G13D 2+ A)Rpeettv Ca Representative (Ai) ngtv el.Teefc of effect The cells. -negative eusrto oCa to sequestration m 2+ T nCa in ATP M lxfo ER-localized from flux 2+ 2+ Clise l,2001; al., et (Collins 2+ G13D paedrn IP during uptake paewudalso would uptake 2+ dltdHKH2 -deleted 2+ ´ 6 fe imaging -free dnse al., et rdenas eeae by generated ...o data of s.e.m. 2+ 2+ responses G13D arising 1611 2+ in 2+ 3 3 - -

Journal of Cell Science ye eeacneuneo lee IP altered of consequence a were types Ca integrated the 6Bii), mitochondria (Fig. responding types cell of a between Although percentage observed 6Bi). remained the was (Fig. this in recording and the difference types, of minor duration cell the both for of elevated mitochondria of majority Ca iohnra irdmiso u oa nrni leainin alteration intrinsic an Ca to due the or microdomains mitochondrial ufrfloigdpeino nrclua RCa ER intracellular of depletion following buffer EERHARTICLE RESEARCH 1612 Ca store-operated with associated Ca mitochondrial significantly the in differences was 6Biii). cells (Fig. cells HKH2 HCT116 in K-Ras-deleted than greater the of response Ca in increase an induced ATP 2008). al., et Szado iohnra Ca mitochondrial nCa in xeiet eete efre odtriewehrthe whether determine to performed then were Experiments 2+ nlxwsiiitdb eadto fCa of re-addition by initiated was influx 2+ 2+ fe mgn ufr(oln ta. 01 Giacomello 2001; al., et (Collins buffer imaging -free paepoete ftemtcodi.T hsend, this To mitochondria. the of properties uptake 2+ paedrn h nraei yooi Ca cytosolic in increase the during uptake 2+ 2+ nlxwsas monitored. also was influx paebtentetocell two the between uptake 3 inligi ER– in signalling 2+ 2+ oteimaging the to trswt Tg with stores 2+ nthe in 2+ 2+ irdmiso ihCa high of microdomains rvosysonta eain nue ppoi through apoptosis induces menadione that shown previously anridpneto natrto nteitiscCa intrinsic the in alteration an of independent manner reported the with consistent cells Ca are HKH2 of observations properties and These HCT116 6C). between (Fig. observed was mitochondria estvt fHT1 n K2clst ppoi nue by Ca induced via apoptosis acts to that cells stimulus HKH2 a and HCT116 of sensitivity ta. 00 asne l,20b.Udrteecniin,a Ca conditions, mitochondria integrated the these responding in difference of Under no percentage and 2008b). observed the al., was in et difference Hanson minor 2010; al., et iohnra Ca mitochondrial mitochondria. the of properties Clise l,20;Gaoel ta. 00 asne al., ER–mitochondrial et Hanson that 2010; indicated al., data et Ca these Giacomello Together 2001; 2008b). al., et (Collins samaueo h ucinlcneune fehne ER– enhanced of consequences functional the of measure a As 2+ lxi nacda euto K-Ras of result a as enhanced is flux ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal 2+ 2+ paefo ukctslrte hnfo the from than rather cytosol bulk from uptake lxfloigK-Ras following flux E,Ei)Rpeettv muolto IP of immunoblot Representative Eiii) (Ei, to (mean respect cells with HCT116 cells HKH2 protein in calreticulin expression in change Fold (Dii) calreticulin. of immunoblot Representative (Di) el ihrsett C16cls eut are Results means cells. HCT116 to respect HKH2 with in cells Fold expression (Cii) SERCA2b control. in loading change a as used (CLNX) was Calnexin SERCA2b. of immunoblot (Student’s B)Epeso fteDE Ca D1ER the analysed. of were Expression coverslip (Bi) per cells 60 least 0DE-xrsigclsprcvrlpwere * coverslip analysed. per cells ( D1ER-expressing day 10 each on imaged cell were per type coverslips three where experiments, i.5 RCa ER 5. Fig. xrsino RCa ER of expression yewr mgdo ahdy( day each on imaged cell were per type coverslips three where experiments, means are oesml Student’s (one-sample sda odn oto.(i,Ev.Fold Eiv). IP (Eii, in control. change loading was a (CLNX) as Calnexin used cells. the in expressed 1Rtasetdcls h Ca The cells. of D1ER-transfected signals FRET in Representative image (Bii) overlay yellow). red; (in calnexin ER-localized protein (in the YFP with of colocalizes excitation green) its of by localization determined and D1ER expression The ER. the eoeldi K-Ras in remodelled Ai ekCa Peak (Aii) loecneo h w eltpsimaged. means types are cell Results two the of fluorescence cells. Bi)Bsln-utatdCa Baseline-subtracted HCT116. (Biii) to in compared higher cells is HKH2 ratio) emission by YFP:CFP (represented the experiment the of beginning ftasgri nCa in application thapsigargin following of cells HKH2 and HCT116 nat IP anti an anti-IP an with detected epc oHT1 el.* cells. HCT116 to respect (Eiv, 2+ 2+ n tteE–iohnra interface ER–mitochondrial the at a sesd eadohr have others and We assessed. was 5 A)Rpeettv Ca Representative (Ai) 6 7 xrsini K2clswith cells HKH2 in expression 27) ...( s.e.m. 3 3atbd Ei) IP (Eiii). antibody R3 6 t P ts) C)Representative (Ci) -test). ...o aafo asof days 4 from data of s.e.m. , 3 2+ 1(Eii, R1 2+ .5 ** 0.05; epne Ai)AC Results AUC. (Aiii) response. n eesaegetrand greater are levels 5 6 1frSERCA2b). for 21 6 ...o asof days 3 of s.e.m. 2+ 2+ n G13D P s.e.m., fe mgn buffer. imaging -free 5 t G13D , hnln rtisis proteins -handling 3 -test). G13D 1atbd E)and (Ei) antibody R1 5 n IP and 15) .1 *** 0.01; dltdHKH2 -deleted P , 2+ 2+ .5 ** 0.05; n 2+ -free eein the deletion, eeini a in deletion 5 3 n 2+ epne in responses 2+ 2wsnot was R2 n 8). 5 epneof response P 5 ee tthe at level esrin sensor ) tleast At 9). , 2+ 3 2.At 12). R3 0.001 P uptake , 0.01 3 Rs

Journal of Cell Science neeaini iohnra Ca mitochondrial in elevation an EERHARTICLE RESEARCH ecieoye pce RS-eedn ciaino IP of activation (ROS)-dependent species oxygen reactive ohHT1 n K2cls(i.7) plcto of Application 7B). (Fig. cells. population cell HKH2 menadione-treated and the cells of HCT116 percentage both the HKH2 However, in apoptosis in and detected induced were menadione (PI) death HCT116 cell iodide of propidium levels cell both Basal with 2008a). flow the the al., stained by et in determined cells (Hanson was of observed of content DNA analysis that percentage cycle. cytometric than cell the lower the of phase content G1 as DNA with assessed population with 50 was at cells cells fragmentation greater HCT116 of a number than greater exhibited treatment the however menadione cytochrome throughout cells to HKH2 diffusely sensitivity 7Ai). distributed cells (Fig. became cytosol HCT116 with treatment and following and cytochrome lost menadione was of distribution HKH2 against distribution mitochondrial mitochondrial this untreated antibodies typical a Although displayed with 7Ai). immunolabelled (Fig. cells cytochrome cytochrome of end, of imaging hallmarks this as Cytochrome used To were apoptosis. fragmentation 2008). DNA and al., mitochondria et Szado c c en otfo h iohnrai significantly a in mitochondria the from lost being olwn xouet eain o 0h 20 for menadione to exposure following c itiuinwsasse yconfocal by assessed was distribution m eain Fg Ai.DNA 7Aii). (Fig. menadione M 2+ Bugrnre l,2009; al., et (Baumgartner c osfrom loss 3 sand Rs c , eemndta K-Ras that determined nHT1 n K2clsb mgn Fg A)and 7Ai) (Fig. imaging by cells S2). HKH2 Fig. material and treatment (supplementary hallmark immunoblotting menadione HCT116 following further observed a in also activation, was apoptosis, Caspase of 7B). (Fig. counterparts ee epoietefrtdmntaino nitrcinof interaction Ca an with of K-Ras demonstration understood. oncogenic first poorly expressed the is natively provide transformation we oncogenic Here, of context the ihDAcnetlwrta nG hs a significantly was phase G1 K-Ras in the than in lower greater content DNA with paeb h iohnracnrbtst h pro-survival the to contributes mitochondria the by uptake Ca on K-Ras oncogenic of impact The DISCUSSION of phenotype oncogenic the to cells. contributes HCT116 mitochondria the to Ca sgncclncne elln ar xrsigete single a K-Ras either expressing mutant pairs of line cell comparing copy By cancer fate. cell colon affect isogenic might crosstalk signalling this how estzsclst r-ppoi tml.Fo hs aa we data, these From IP of stimuli. suppression pro-apoptotic that propose to cells sensitizes ae oehr hs aaidct htdcesdfu fCa of flux decreased that indicate data these together, Taken 2+ inl n RmtcodilCa ER–mitochondrial and signals ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal G13D xeiet r nCa in are Experiments infcn (Student’s significant epne uigsoeoeae Ca operated store during responses Ca Mitochondrial 6. Fig. C)Rpeettv iohnra Ca mitochondrial Representative (Ci) Ci)ACo iohnra Ca mitochondrial of AUC (Ciii) A ersnaieiae fclsexhibiting Ca cells mitochondrial of images Representative (A) 100 a.A es e el e oesi n tleast analysed. at were * and cell per coverslip per mitochondria five cells each ten on least imaged At were day. type three cell where per experiments, coverslips of days 3 means from the represent graphs bar K-Ras of loss by enhanced iohnra Ca mitochondrial Scale indicated. 10 are bar: experiments of points Time Bi)ACo iohnra Ca mitochondrial of AUC mitochondria. (Biii) responding of Percentage (Bii) hc a ntae yadto fCa of addition by initiated was which trswt g(ro) Ci ecnaeof Ca Percentage exhibiting (Cii) mitochondria (arrow). Tg intracellular with of stores depletion following buffer imaging P G13D , G13D .5 ** 0.05; dltdclsta nterHCT116 their in than cells -deleted m T nteasneo xrclua Ca extracellular of absence the in ATP M m 3 eeinehne IP enhances deletion .(i ersnaietae of traces Representative (Bi) m. rn uatKRsw have we K-Ras mutant no or inligfo h RadCa and ER the from signalling P , .1 *** 0.01; 2+ 2+ epne nclsepsdto exposed cells in responses 2+ epne o100 to responses t -test). 2+ 2+ inl,priual in particularly signals, P fe mgn buffer. imaging -free , 2+ lxadta this that and flux 2+ .0;ns,not n.s., 0.001; 2+ G13D epne are responses responses. 2+ 2+ inligand signalling 6 response. epne All response. nHH cells. HKH2 in ...o data of s.e.m. 3 2+ -dependent 2+ 2+ m entry, ATP. M othe to 1613 2+ 2+ 2+ .

Journal of Cell Science lsammrn,Ca membrane, plasma ota ta. 03.Truhmnplto fteeCa these of manipulation Through 2000; 2003). al., et al., Berridge 2003; et al., et Bootman (Berridge both of combination 00 ohd ta. 02.Atrdepeso fanme of number al., a et of Szatkowski expression 2009; Altered al., 2012). Ca al., et et Lipskaia Yoshida 2001; death 2010; al., and et migration (Cre Legrand described proliferation, been cell have of regulation including EERHARTICLE RESEARCH acrlaigt h rpslta lee Ca altered that colon proposal 1614 with the patients gene in to not the identified leading has in been cancer mutations causality have somatic SERCA cells, encoding However, transformed demonstrated. in been pathways signalling each for roles specific Ca cells, these of transformed in pathways signalling K-Ras of properties idnsaesgetv fa motn oeo eti Ca certain of these role Although important 2013). an al., of et suggestive Motiani are 2007; 2012; al., findings al., et Monteith et 2006; al., et Monteith Korosec 2012; al., et (Arbabian phenotype. yooi Ca Cytosolic 2+ hnln rtisi uortsu a lobe determined been also has tissue tumour in proteins -handling 2+ ore ncnrligapcso acrcl biology cell cancer of aspects controlling in sources 2+ inl r eeae yCa by generated are signals G13D 2+ ´ i ta. 07 ue ta. 2004; al., et Humez 2007; al., et pin ees rmitaellrsoe ra or stores intracellular from release soitdwt h oncogenic the with associated 2+ nr costhe across entry 2+ signalling 2+ 2+ yhmlgu eobnto Siaaae l,19) By 1993). al., et deleted (Shirasawa been a has and recombination oncogene oncogene driving homologous the activating which cell an by in cancer of line a allele cell upon isogenic single second performed al., of a are use et studies harbouring the which Tuveson line in through 1997; pairs minimized line al., are cell et issues These the (Serrano on 2004). overexpression depending K- proliferation oncogenic of cell because or level concern senescence essential particular induce also can a activated Ras is is of this effects oncogene the isoforms, analysing driving Ras For 2008). the for Cook, and expressing controls (Roderick appropriate line of cell availability The the cells. live in possible inligptwy sta h nlsso Ca of analysis the that is pathways signalling 00.Hwtentv xrsino pcfcocpoenin oncoprotein Ca specific a affects of cells expression native transformed the Weinberg, and How activity (Hanahan respectively the 2000). encode, suppress they or proteins promote as the to of such serve oncogenes that 2006). in suppressors al., tumour mutations et through (Korosec arise transformation Cancers oncogenic to predisposes otiue otepeoyeo h rnfre elis, cell transformed Ca investigating the when issue of particular phenotype A clear. the not however, to contributes el ihaDAcnetlwrta nteG hs (Sub-G phase G1 HKH2 the in and HCT116 than lower of content percentage DNA the a upon with h cells 20 for menadione to oprsnwt C16( HCT116 with comparison fHH el ipae ifs cytochrome diffuse displayed percentage higher cells a HKH2 menadione, of with treatment Upon 50 c. to cytochrome exposure 10 h bar: 20 Scale following menadione. cells HKH2 and HCT116 i.7 estvt oaotssidcdb eain is K-Ras menadione of by loss induced by apoptosis enhanced to Sensitivity 7. Fig. mgso cytochrome of images ANOVA). otn a esrdb lwctmti nlsso PI-stained of means analysis are cytometric Results flow DNA. by measured was content ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal c gen n ciae aps rd in (red) 3 caspase activated and (green) 6 m n ...( s.e.m. .(i)Nme fclsehbtn diffuse exhibiting cells of Number (Aii) m. 5 2+ G13D xeiet) B feto exposure of Effect (B) experiments). 3 inligadwehrthis whether and signalling nHH cells. HKH2 in n 5 ) * 5). P , c 2+ .5(two-way 0.05 itiuinin distribution inligi only is signalling A)Confocal (Ai) m M KRAS 1 .DNA ). or 2+

Journal of Cell Science uhpiso ellns efudta oso K-Ras of loss that found we lines, cell of pairs such ta. 00.A nraei IP (Szatkowski in estradiol increase with An 2010). proliferate al., to et induced cells MCF-7 h xrse IP expressed the a nlee yK-Ras by unaltered was nclrca acrcl ie,K-Ras lines, cell cancer colorectal in K-Ras in particular, In cells. nte PR eeas rae nK-Ras in greater also were GPCR, another a nrae n IP and increased was -mnehxdpey oae(-P)idcdapoptosis IP induced 2003). al., (2-APB) et (Sakakura borate 2-aminoethoxydiphenyl aebe eotdpeiul.Ms oal,a nraein increase an notably, Most previously. IP reported been have yooi Ca cytosolic ubro ehnss o xml,b nrae GPCR IP in increased changes in by metabolism, modification effectors, phosphate example, downstream inositol to For coupling or mechanisms. expression of number Ca niaigta Ca that indicating 03.I ati acrcls nices nIP in increase an cells, cancer gastric al., et In (Vartanian lines 2013). cells K-Ras-deleted of analysis microarray el Hiha ta. 96.Gvntedfeigpoete of properties differing (NSCLC) the cancer Given lung 1996). IP cell al., each non-small et in (Heighway observed cells been has Ras bnac ugssta nacdbslsgaln possibly signalling Ca basal of enhanced PLC that involving suggests abundance nieyta oiiain nprnri eetrexpression receptor K-Ras of purinergic effects in the to modifications contributed that unlikely inl eegetri w ifrn ooetlcne ellnsin lines cell cancer K-Ras colorectal different which two in greater were signals Ca hormone-agonist-induced comparing ARTICLE RESEARCH epne nue yacl-emataaou fIP of analogue cell-permeant a by induced responses K-Ras to response common a is signalling Ca aue eseuae htRscuditreewt n ralof all or IP any and activity with PLC interfere Increased could processes. Ras these that speculated we nature, rae nK-Ras in greater xrsin hne nteepeso fIP of expression the in Changes expression. h Ri K-Ras in ER the Ca the of properties the defining -a ih loices inligatvt hog stimulating through activity breast, signalling PLC Oncogenic in increase 2005). also (Weber, and might transformation K-Ras of lines is result signalling cell a basal as that transformed enhanced suggesting of carcinoma, colonic number and ovarian a in reported Ca greater a through bevdi h sie,btnti acrclsestablished IP cells ascites, cancer the in In not tumours. but ascites, primary the from in observed elln.A hscl-ematIP cell-permeant this As line. cell Ras lines. Ca icmetn PR,Gpoen n L,ordt niaethat K-Ras indicate of data levels our PLC, native and proteins G GPCRs, circumventing by reduced was Ca expression hormone-stimulated of K-Ras suppression when that cells indicating siRNA, HCT116 in as -a eeinmgthv motn osqecs sdifferent As consequences. important IP have might deletion K-Ras bevtosta Ca that observations 3 3 h rae antd fhroeidcdCa hormone-induced of magnitude greater The oal,atog h obndepeso fIP of expression combined the although Notably, 2+ 2+ 2+ sfrsaergltddfeetyb IP by differently regulated are isoforms R recent a in detected been has level mRNA the at expression R3 G13D e ees rmteER. the from release hc ntr,b nraigIP increasing by turn, in which , inl nue ysiuaino h itmn receptor, histamine the of stimulation by induced signals 2+ ees rmsoe Kle ta. 01.Hwvr our However, 2001). al., et (Kelley stores from release 3 dltdclspritdi Ca in persisted cells -deleted sfr,ti hnei sfr opsto following composition isoform in change this isoform, R inligi el abuigmttdK-Ras mutated harbouring cells in signalling G13D 2+ e 3 inl.Teeeae Ca elevated The signals. G13D osntcnrbt oRsmdae regulation Ras-mediated to contribute not does utpswsatrdi h K-Ras the in altered was subtypes R G13D a endltd(K2adDO) swell as DKO4), and (HKH2 deleted been had 2+ 2+ 2+ dltdclsta nteioei parental isogenic the in than cells -deleted 3 dltdclscudhv rsntruha through arisen have could cells -deleted G13D 1epeso upesd niaigthat indicating suppressed, expression R1 ees rmteE a motn in important was ER the from release inl r eaieycreae ihRas with correlated negatively are signals otn fteE.Gvnispleiotropic its Given ER. the of content G13D 3 a ietefc nIP on effect direct a has G13D 3epeso sas nrae in increased also is expression R3 eein h eaieaudneof abundance relative the deletion, 3 2epeso oehrwt K- with together expression R2 dltdcls IP cells, -deleted 2+ G13D epne motnl,Ca Importantly, response. 3 ietyatvtsIP activates directly 3 eein iial,Ca Similarly, deletion. 2+ 2+ G13D ees ol promote would levels, G13D fe mgn buffer, imaging -free G13D 2+ inligbetween signalling 3 3 3 si acrcells cancer in Rs 3ihbto by inhibition R3 3 dltdcls tis it cells, -deleted 3 epne nK- in responses n Ca and nclrca cell colorectal in erse IP represses eeshv been have levels or expression R 2+ 3 G13D 3expression R3 G13D 3 from release 3 R-mediated isoforms R 2+ 3 enhanced G13D 3was R3 -deleted giving , 3 were .As 3 3 Rs, R3 2+ 2+ 2+ usatal oteehneeti Ca in enhancement the to substantially oiatdtriato h antd fteIP the of magnitude the of determinant dominant rbbyalwsgaln rmec eetrt atcpt in participate to Ca receptor choices. fate each cell from different signalling allow probably rmIP from Ras K-Ras elto,adicessRSadE tes(reise al., et ER (Orrenius lower of stress benefit the ER of ATP Ca view and well-accepted cellular this ROS Despite to 2003). increases leads and exposure, depletion, phosphatidylserine promotes htti leaini RCa ER in alteration this that Pno ta. 08 oeikadCo,20) yincreasing By 2008). Cook, IP and the Roderick through 2008; leak al., et (Pinton ebr lordc IP reduce also members Hgre l,19) sarsl,weesepeso fIP of expression whereas result, a As 1998). al., et (Hagar Sakwk ta. 00.TeedfeetCa different profile These oscillatory more 2010). a al., to et peak–plateau a (Szatkowski from responses feature notable A types. IP cell of two the between observed signalling nlsdi C16cls opoietrl o a in Bax that for hypothesis the supporting role 2004), was al., prominent Ca et (MOMP) Marchi no permeabilization (De detected membrane cells, outer HCT116 when mitochondrial Notably, and shown). in HCT116 not (data the analysed here between analysed detected types Bax, Bak, cell was of HKH-2 Mcl-1 expression or the in Bcl-XL differences Bcl-2, No 2005). al., et Oakes et Bakhwe l,20;Ka ta. 96 edse al., these et of outcome Mendes potential 1996; Ca a al., Thus, different 2010). et al., Khan et Szatkowski 2000; 2005; al., et (Blackshaw death htarltvl o ee fCa of level low relatively a that h rnfre el(ege n ue,20) pcfcly by Specifically, 2008). Huber, Ca and limiting (Bergner cell transformed the hra h euto nIP in reduction the whereas h niaottcfml ebr)rsl nalwrn ffree of lowering enhanced a in of example, Ca result abundance members) ER BH3-only increased For family of in anti-apoptotic knockout result the cancer. or (which members proteins in family pro-apoptotic Bcl-2 pathways dysregulated of death expression of cell are of mechanism regulation the in that into involved proteins investigation of through actions emerged has data Ca iaaae l,19) h hnei h eaieaudneof abundance relative the in change IP the each 1999), al., et Miyakawa ih eotmzdfrcnrligctknss(ite tal., the et whereas 1998), (Kittler al., Ca et cytokinesis (Dolmetsch sustained expression controlling gene for and 2004) optimized be might Ca regular supports Ca hc eut npreblt rniinadatvto of Cook, and activation Roderick Ca and 2008; al., Elevated transition et permeability 2008). (Pinton pathways in apoptotic mitochondria, results in accumulation through which mediated are ER the from C16clsde o nov leain nteepeso of IP expression the members. in alterations family involve not Bcl-2 does cells HCT116 bevdi IP in observed hshrlto yteposria iaePBAt(Khan PKB/Akt kinase pro-survival the by phosphorylation ppoi sdmnse.Tecttxcefcso Ca of effects cytotoxic The diminished. is apoptosis ta. 04 iaaae l,19) ned iN euto in reduction siRNA Indeed, 1999). al., et IP Miyakawa 2004; al., et iet itntCa distinct to rise 3 nices nE Ca ER in increase An 2+ 2+ 2+ 2+ 3epeso nMF7bes acrclstasomdCa transformed cells cancer breast MCF-7 in expression R3 G13D 3 rnin Brig,20;Crpoe l,20) ti likely is it 2003), al., et Caroppo 2006; (Berridge, transient signals. o uvvlo rnfre el,mc ftesupporting the of much cells, transformed of survival for inligrmdligb noei -a ciainin activation K-Ras oncogenic by remodelling signalling 3i hti slatsniiet Ca to sensitive least is it that is R3 G13D 2+ ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal 3 3 dltdcls steCa the As cells. -deleted sfr ol otiuet h ifrne nCa in differences the to contribute could isoform R 1i C16clsssanterrpdproliferation, rapid their sustain cells HCT116 in R1 eesadrdcdfu fCa of flux reduced and levels dltdcls u aaaecnitn ihteview the with consistent are data Our cells. -deleted 2+ 2+ 2+ 3 ees,teefc fsiuita ev oinduce to serve that stimuli of effect the release, intrsi htteCa the that is signatures 3epesn el Amrae l,21;Hattori 2010; al., et (Almirza cells R3-expressing inl rsn rmIP from arising signals 2+ 2+ 3 rsprsigteratvt,Bl2family Bcl-2 activity, their suppressing or R inligfnepit Htoie l,2004; al., et (Hattori fingerprints signalling siltos oohscCa monophasic oscillations, 2+ 3 2+ idcdCa -induced loatvtsDAendonucleases, DNA activates also tr otn a loosre nK- in observed also was content store 3 3poet h el rmcytotoxic from cells the protects R3 3 2+ ciiyi lordcdby reduced also is activity R 2+ siltosaiigfo IP from arising oscillations nteE fesa datg to advantage an offers ER the in 2+ 2+ 2+ tr otn contributes content store ees Ce ta. 2004; al., et (Chen release otn fteE sa is ER the of content 2+ 3 2+ 3mgtpooecell promote might R3 2+ 2+ inligosre in observed signalling siltosta arise that oscillations dpnetinhibition -dependent otemitochondria the to 2+ 2+ rninsare transients 3 -stimulated signatures 2+ release 1615 3 3 R1 R1 2+ 2+

Journal of Cell Science hog hspeerdptwy iohnra function, is death, mitochondrial cell apoptotic IP pathway, by of modulated induction acutely and preferred metabolism including this Through Hajno 2010; rmteE Rzuoe l,21) iohnra Ca Mitochondrial 2012). al., et (Rizzuto ER the from nefc ontdiemtcodilCa mitochondrial drive not do interface olwn K-Ras following rmteE Ksie l,20;Okse l,20) positive A 2005). al., et Oakes Ca 2006; ER al., between et correlation (Kasri ER the from rlfrto n nue eldahi rnfre el,it cells, transformed Ca absolute in the death correlate to cell important be induces would and proliferation K2clsidctsta h xrsino K-Ras of expression the that indicates cells HKH2 Ca high of microdomains such, eueti lxi C16cls nln ihterlwrER– lower their with line In cells. HCT116 Ca in mitochondrial flux this reduce h is vdneo oiiain nmtcodilCa mitochondrial in modifications of evidence first the iohnrai o etitdt h IP the to restricted not is mitochondria iohnrafloigCa following mitochondria 07.I iewt hs tde,SRAbepeso was expression SERCA2b studies, these K-Ras with in increased (Hovnanian, significantly line phenotype In Darrier’s skin In in a 2005). by results 2007). characterized allele al., is SERCA et which one disease, cancers of (Prasad tumours cell loss mice however, squamous in humans, expression of haploinsufficient of induction SERCA detected SERCA2b loss the of in by been in importance demonstrated The result also also 2007). is that have al., SERCA et activity (Monteith in lines or Mutations cell cancer-derived expression tumours. in observed in been and has pump SERCA of euto nSRAbatvt otiue otepeoyeof Ca phenotype ER the in increase to The contributes cells. activity K-Ras-transformed SERCA2b in reduction oee,bcuedpeino h RCa 2001). ER al., the et of (Legrand lines depletion cell because cancer However, prostate in observed IR ossetwt nrae IR hsehne ER-to- enhanced this IICR, increased Ca with mitochondria Consistent IICR. ta. 06 acie l,21;Saoe l,20) iaethat kinase cancers. a many 2008); in al., et activity Szado in 2012; increased al., is et Marchi 2006; al., et ARTICLE RESEARCH 1616 Ca ER-to-mitochondria the in increase pteilcls(ege ta. 09.Cnitn ihthe with Consistent 2009). Ca al., ER et in (Bergner bronchial reduction human normal cells to compared epithelial when lines cell cancer lung aldmtcodilascae ebae MM) hc are Ca which in enriched (MAMs), membranes mitochondrial-associated called eee el ih lob xlie yteosre euto in reduction observed the by explained IP be also might cells deleted nrae estvt fteeclst Ca to cells these of sensitivity increased hc a enpooe oseiial eit pro-apoptotic mediate specifically to proposed Ca been has which ontemo noeosocgncKRs h bainof ablation The K-Ras. K-Ras oncogenic oncogenic endogenous of downstream a nlsd ne hs odtos Ca conditions, these Under analysed. was Radcl rlfrto nteestudies. these in proliferation cell and ER Bakhwe l,20;Ka ta. 96 edse l,2005). al., et Mendes 1996; al., et Khan 2000; al., et (Blackshaw ifrnebtenteHT1 n K2clswsls when lost was cells HKH2 and Ca HCT116 the between difference o-fiiypoes ssc,mtcodi preferentially mitochondria such, As Ca process. accumulate low-affinity a ugs htteehneeto RmtcodilCa ER–mitochondrial and of Rizzuto enhancement therefore data 2002; the mitochondrial Our al., that 2008). the Duchen, suggest et of and Szabadkai (Collins properties 2006; the Pozzan, alone to mechanisms due uptake is observed uptake 3 eraei RCa ER in decrease A h iohnraaea motn agto Ca of target important an are mitochondria The 2+ 2+ 1 hc a enrpre ocnrbt oteCa the to contribute to reported been has which R1, paedrn Ca during uptake lxsa h A,i lordcdi C16cells HCT116 in reduced also is MAM, the at fluxes ´ zyadCsorda and czky 2+ 2+ ees hnesicuigIP including channels release G13D 2+ 2+ G13D tstso ls poiinwt h ER the with apposition close of sites at lxadaotss h xrsino IP of expression the apoptosis, and flux lxi K2clswsmroe yan by mirrored was cells HKH2 in flux 2+ nrae h cuuaino Ca of accumulation the increased 2+ 2+ baini hog oiiainof modification through is ablation 3 ncne,ardcini expression in reduction a cancer, in mdae Ca -mediated otn a endtce nasbe of subset a in detected been has content 2+ nlxfo h xrclua space extracellular the from influx 2+ ees rmteE.Ntby the Notably, ER. the from release ´ eesadpoieainhsbeen has proliferation and levels ,21;Rzuoe l,2004). al., et Rizzuto 2010; s, G13D 2+ dltdcls hwn hta that showing cells, -deleted tteER–mitochondrial the at 2+ 3 2+ -otiigMM.As MAMs. R-containing 2+ 2+ idcdcl et.The death. cell -induced inl.Hr,w show we Here, signals. eusrto n the and sequestration rnfrosre in observed transfer 2+ 3 s(Csorda Rs 2+ 2+ 2+ paeit the into uptake nK-Ras in otn fthe of content tr inhibits store G13D 2+ 2+ 2+ paeis uptake 2+ released ´ 2+ 2+ tal., et s csto acts uptake nthe in G13D 3 leak flux R3, - etrdtesniiiyt Ca to sensitivity the restored sgncmdl niae htsprsino Ca of suppression that indicates models isogenic 09%cnlec.Cvrlp eecae ihpoly- with coated were Coverslips confluency. 80–90% eusrto n eldaha euto oso K-Ras of loss of result a as death cell and sequestration H and Menadione u oK-Ras to due tafnlcnetaino 5n.Temdu vryn h el was cells the Ca and overlying measured medium expression protein The and nM. h, 25 24 after of exchanged concentration final a at tteE,hsbe eotdt pcfclymdaethe mediate specifically to reported been IP has localized of is ER, which dephosphorylation PML, the suppressor tumour at the recently, More yooi Ca Cytosolic Ca cytosolic of Imaging Thermo) (Dharmacon, 2 Briefly, reagent instructions. manufacturer’s transfection the to non-targeting according Dharmafect-2 siGENOME transfected reverse and were using Thermo) K-Ras (Dharmacon, for oligonucleotides control Pool SMART siGENOME transfection siRNA K-Ras (both cells DLD-1 and HCT116 culture Cell METHODS AND MATERIALS ouainb K-Ras by modulation omnrsos oK-Ras to response common oal,teritouto fE-agtdPLi PML in PML ER-targeted of reintroduction the Notably, rmr as,sc smttosi GRo -a,rie the raises B-Raf, or the EGFR not Ca in of modulation is mutations the that Ras possibility as mutated such where cause, cancers primary and activation in Ras pathways of downstream importance The or metabolism. proliferation of cell modulation of promote to enhancement serve including that transformation physiology cell cell of aspects other to contribute 03.Bify el eesee nopoly- onto seeded were cells Briefly, 2003). ooetlcls h nacmn fCa of enhancement The cells. colorectal 2wl iho 4 or dish 12-well xeiet efre fe ute 4h. 24 further a after performed experiments (FBS) penicillin, serum units (5 bovine solution fetal penicillin/streptomycin 55 heat-inactivated CA, 1% Dulbecco’s Carlsbad, 10% in (Invitrogen), Technologies, cultured containing Life were (DMEM; USA), Cells medium 1993). Eagle’s al., modified previously et been have (Shirasawa and Japan) described University, (Fukuoka Shirasawa Senji of nCa in he oesisprcl yewr mgdprdyo three on day per imaged were captured and were type mirror Images cell camera. dichroic filter. (CCD) per nm device pass 400 charge-coupled and coverslips long ER ms, a ORCA 200 Three a nm of Hamamatsu 460 using at exposure a a an using selected USA) with through CA, s was 3 a filtered Novato, every using light inverted pair selected Industries, image was emitted TE200 1 nm (Sutter 380 of Thames, wheel Eclipse and frequency Upon 340 20 filter Nikon Kingston at PlanFluor light (Nikon, motorized a Excitation Nikon objective UK). of immersion a Surrey, multi with stage NA Coverslips equipped 0.75 the min). microscope 30 on further epifluorescence and a imaged for chambers buffer were imaging imaging 2 experiment, in steel Technologies; each de-esterification (Life by to stainless AM Prior fura-2 into with h. loaded mounted 48 after were imaged coverslips and densities equivalent agtfrintervention. for target a 00.Ti idn scnitn ihteicesdsniiiyto K-Ras sensitivity increased the the in al., with observed et consistent menadione (Giorgi is finding Etoposide) This agent 2010). DNA-damaging the by (induced edn fcells. of seeding ne %CO 5% under sgncdrvtvsHH n K4(ohK-Ras (both DKO4 and HKH2 derivatives isogenic ncnlso,ordt ecie o h is ietealterations the time first the for describes data our conclusion, In m tetmcn Sga ost K.Clswr ananda 37 at maintained were Cells UK). Dorset, (Sigma, streptomycin) g 2+ ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal euaindie yasnl noei K-Ras oncogenic single a by driven regulation 2+ 2 G13D nstrtdhmdt n eepsae pnreaching upon passaged were and humidity saturated in 6 a mgda rvosydsrbd(epate al., et (Peppiatt described previously as imaged was 10 2 sacmo etr fmn acr n sthus is and cancers many of feature common a is O 5 el na6wl ihwr rnfce ihsiRNAs with transfected were dish 6-well a in cells 2 ,btntta oCa to that not but ), G13D 2+ 3 3b Pa(irie l,2010). al., et (Giorgi PP2a by R3 G13D nclrca el slkl loto also likely is cells colorectal in 2+ dpnetaotss(nue by (induced apoptosis -dependent wn oispeorpcactions, pleiotropic its to Owing . G13D 2+ G13D/WT lxsosre nti study this in observed fluxes altdHH cells. HKH2 -ablated 2+ L 2+ lsn-otdcvrlp at coverslips -lysine-coated adig mitochondrial handling, m idpnetapoptosis -independent o 0mn followed min, 30 for M n hi respective their and ) -/WT 2+ eeakn gift kind a were ) L 6 inligi a is signalling lsn ro to prior -lysine G13D 10 G13D 5 2+ 2 el na in cells leein allele / imaging 2 ntwo in cells 6 ˚ C /

Journal of Cell Science yceiuiecne(C)(hroSinii)adsubsequent (Life and antibodies secondary Scientific) fluorescently-labelled and All (Thermo of detected film, one to were 1:5000). (ECL) and exposure antibodies TBS-T at chemiluminescence HRP-conjugated of detection. both exchanges by band five LI-COR; before in washed TBS and then were Technologies membranes (Life secondary (Jackson fluorescently-labelled (HRP)-conjugated antibodies or dilution) peroxidase 1:10,000 horseradish then Immunoresearch; were either Membranes with TBS-T. with probed washing by removed were antibodies anti-IP fFakWyak(utc ta. 94] anti-IP 1994)]; al., et (Wuytack gift Wuytack 1:1000, [dilution Frank anti-SERCA3 of Sigma); 1:20,000, (dilution calnexin ocnrto a acltdacrigt rniwc tal. Ca et D1ER Ca genetically-encoded FRET-based, cells. Grynkiewicz The Ca ER 50 to of Imaging least according at 1985). al., calculated containing et (Grynkiewicz coverslip was each concentration days, separate ARTICLE RESEARCH neuvln muto ahsml 1 o50 to Scientific). (15 (Thermo sample kit each quantified assay of amount was protein equivalent lysate (BCA) An protein acid and bicinchoninic seeding a post h using 48 harvested were Cells Immunoblotting Ca Mitochondrial Ca mitochondrial of Imaging ihpiayatbde dltda niae eo nTSTml)for milk) 4 or TBS-T temperature in room below at 0.05% indicated probed h containing as subsequently 1 TBS (diluted were in antibodies Membranes h milk. primary 1 5% with and for (TBS-T) incubation 20 by Tween blocked first protein-binding were Non-specific nitrocellulose. sites or controls) loading their and dlto :00 idgf fFakWyak nvriyo Leuven of University Wuytack, anti-IP Frank 1989)]; of al., gift et anti-SERCA2b (Wuytack kind AbdSerotec); 1:500, 1:1000, (dilution [dilution anti-K-Ras follows: as are otoldb h iiehVxelSa otae Ca camera software. Scan CCD Voxcell ER argon/krypton Visitech band-pass ORCA the an Hamamatsu nm by a of 575/50 controlled using a line captured through were nm Images filtered microscope 568 filter. was the TE2000 fluorescence with Scan Emitted Nikon illumination laser. VoxCell by a a VisiTech 60 excited for on Nikon was a a configured using buffer with imaged confocal equipped imaging (4 were disc in AM Cells spinning de-esterification min). rhod-2 30 by further with followed loaded temperature were cells oeike l,19) niatv aps 110,B BioSciences); BD (1:1000, anti- 3 and (1:1000; Sigma) caspase anti-calreticulin (1:5000, anti-active anti-GAPDH 2009)]; 1997); al., al., et et Roderick (Harzheim generated in-house Ma Ca a as AM msino F frhrflee hog 3/0n adps filter). the band-pass nm passed 535/30 and a filter), through filtered band-pass (further nm YFP 485/40 CFP of a of a emission fluorescence with through emitted configured Graveney the filtered was reflected a Limited, (further which Emitted unit mirror, using Research splitter dichroic nm image captured (Cairn 515 The UK). simultaneously splitter Kent). was Faversham image Road, Southend-on-Sea, II YFP Optosplit and inverted Cairn (Olympus, CFP IX81 of Olympus Polychrome fluorescence a monochromator using an selected 20 was of steel nm UPlanSApo V 435/10 stainless Olympus stage at light in an Excitation the objective. mounted with on were equipped imaged Coverslips microscope imaged transfection. and were post the Cells chambers Ilkirch, h specification. with Transfection, 24 manufacturer’s (PolyPlus transfected at JetPei to but using according imaging h France) 24 ratiometric after for construct D1ER indicated as seeded ucsfluet oio etn n yai hne nE luminal ER in changes dynamic and resting monitor [Ca to use successful ito m amr(nvriyo ooao ole,UA.Teaffinity Ca The for USA). indicator Boulder, Colorado, the of of (University Palmer Amy of gift acltda rvosydsrbd(oln ta. 2001). al., et (Collins described previously as calculated NPG;Lf ehoois.Poen eetaserdfo h gels IP the (for gels membranes from pre-cast (PVDF) transferred fluoride gradient polyvinilidene were onto Proteins 4–12% Technologies). onto Life or (NUPAGE; gels polyacrylamide self-poured 2+ Ple ta. 04.I xeiet novn 1R el were cells D1ER, involving experiments In 2004). al., et (Palmer ] 3 1[iuin110,i-os eeae Ksie l,20);anti- 2004)]; al., et (Kasri generated in-house 1:1000, [dilution R1 2+ niao Saoe l,20) ro oec experiment, each to Prior 2008). al., et (Szado indicator 2+ 2+ a mgda rvosydsrbduigrhod-2 using described previously as imaged was 2+ a endtrie ob 60 be to determined been has 6 12 Aolimrinojcie Rhod-2 objective. immersion oil NA /1.25 ˚ vrih.Dtiso rmr antibodies primary of Details overnight. C 2+ 3 3(iuin110,B Biosciences); BD 1:1000, (dilution R3 b ci 150,ACm.Excess AbCam). (1:5000, actin m o 0mna room at min 30 for M m 2+ )wslae no7% onto loaded was g) niao a kind a was indicator 3 2+ 2[iuin1:500, [dilution R2 ocnrto was concentration 3 m sadSERCAs and Rs 6 ,alwn its allowing M, 07 Aair NA /0.75 2+ xiaino h ys(lmu V00cnoa ofgrdo an on configured confocal 60 a FV1000 using Vectashield microscope (Olympus inverted for dyes in IX81 lines Olympus the mounted laser imaged appropriate of were were using was excitation Cells microscopy Coverslips nuclei. confocal PBS antibody counterstained point-scanning PBS. in also by Excess X-100 which in Triton DAPI, temperature. washes 0.1% containing in room two washes at five and with h removed 1 PBS, subsequently in for X-100 (Life Triton antibodies Technologies) 0.1% secondary Alexa-Fluor-labelled in with washing After incubated were by Sigma). cells antibodies (1:500, excess anti-calnexin of BioSciences); removal BD (1:200, 3 caspase mgswr rcse n nlsduigIaeJ. Image using confocal analysed and A1R processed Nikon were 60 cytochrome Images using of a and imaging microscope for and inverted objective imaging, immersion Ti Nikon YFP a on and configured calnexin for objective omlzto,poenaudnei h xeietlcniin of conditions experimental control. the the were to Following interest normalized in was band. of abundance interest control protein protein loading the corresponding normalization, of Image the Bands or against detection) detection). normalized ECL LI-COR for (for (ImageJ, either film Studio obtained digitized were of (LI-COR values analysis scanning intensity through digital quantification, band by For detected Odyssey). were LI-COR) and Technologies ihnfrassac ihsaitclaayi,Rce akrfrfo cytometry flow for imaging. Walker for Segonds- Rachel Walker Anne analysis, Simon acknowledge statistical and to with for like assistance Leuven also for KU would Pichon Wuytack, We Frank antibodies. and construct SERCA FRET of the D1ER University Palmer, the Amy for lines, USA cell Colorado, the for Japan University, Fukuoka Shirasawa, omtmeaue eal fpiayatbde r sflos anti- follows: as are at antibodies h 1 primary for of were solution) cytochrome cells Details blocking PBS), in temperature. solution in (diluted blocking room diluted antibodies in serum primary goat incubation with donkey After probed 5% PBS. X-100, in Triton X-100 (0.1% buffer fixation Triton permeabilized with 0.2% and fixed PBS) with were in cells glutaraldehyde seeding, 0.05% post paraformaldehyde, h (2% 48 at al., Briefly, et 2009). (Higazi described previously as performed was Immunofluorescence Immunofluorescence ...adHLR ocie ftesuyaditrrtddt.CP,SJC and S.J.C. C.P., data. interpreted and manuscript. study the the M.D.B., prepared C.P., of H.L.R. data. conceived analysed H.L.R. and and experiments S.J.C. performed H.L.R. and T.C.F. C.P., contributions Author interests. competing no declare authors The interests Competing IP the for Oxford of University Conway, Stuart to grateful are We Acknowledgements Student’s one-sample a control normalized to t compared was data Where or analysis analysis Statistical cytometric after flow h for 20 processed harvested were and immunofluorescence. Cells menadione 100 analysed. to also and exposure culture were 25 in parallel diluted in between menadione cultured to concentrations exposed were at cells seeding, medium post h 24 At cells of treatment Menadione 2008a). al., et a (Hanson through laser filter fluorescence argon emitted pass an of band by intensity nm 585/42 the 488 to at according were excited analysed were cells and suspension (Oxford, Stained in cytometer cells flow (PI). Single FACSCalibur UK). RNase iodide Dickinson that to Becton propidium prior a cells ethanol with with analysed 70% with staining with fixed pooled and were then treatment cells trypsinization. PBS, by and in harvested washing were collected that After substrate were their to medium attached remained the in Cells analysis cycle Cell rtowyAOA infcnewsacpe at accepted was Significance ANOVA. two-way or ts a mlyd te xeiet eeaaye yStudent’s by analysed were experiments Other employed. was -test ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal c dlto :0,SnaCu itcnlg) n anti-active and Biotechnology); Cruz Santa 1:200, (dilution 6 c 13 Aolimmersion oil NA /1.35 n ciae caspase). activated and m .Cnrlsamples Control M. P , 0.05. 3 M Senji BM, 6 14N oil NA /1.4 1617 t -test

Journal of Cell Science uny .D n aa,M. Katan, and D. T. Bunney, oln,T . erde .J,Lp,P n ota,M D. M. Bootman, and P. Lipp, J., M. Berridge, J., T. Collins, ruad .P,Ge P., J. Brouland, D. M. Bootman, and P. Lipp, J., M. P. Berridge, Lipp, and D. M. Bootman, J., M. Berridge, M. R. Huber, and A. Tufman, J., Kellner, A., Bergner, oln,T . ip . erde .J n ota,M D. M. Bootman, and J. M. Berridge, P., Lipp, J., T. Collins, Bootman, L., H. Roderick, S., K. McColl, F., Zhong, I., Valencia, R., Chen, S. Curci, L., Debellis, A., DeLuisi, A., Colasuonno, M., Colella, R., Caroppo, Ca oit uigtecus fti ok eoie nPCfrimmediate for PMC in Royal Deposited the work. from this Fellowship of Research Programme course release. a Training the by Doctoral during supported BBSRC Society also Placement Cambridge was Experience of H.L.R. Research DTP. University a the by Strategic from funded (Institute grant was ISPGs T.C.F. Research the Signalling Grant)]. Sciences by and Programme Biological supported [Epigenetics and are (BBSRC) College, Biotechnology laboratories Council John’s the S.J.C. St and Institute, of H.L.R. Babraham Studentship The Aldobrandini UK. the Cambridge, by funded was C.P. Funding ARTICLE RESEARCH 1618 Edward, and J. M. Berridge, R., O’Connor, L., H. Roderick, D., M. Bootman, F. Khan, McCormick, and and H. S. G. Snyder, Bollag, A., L. C. Ross, H. H., A. Roderick, Sharp, A., and Sawa, S., D. Blackshaw, M. Bootman, J., M. Berridge, J. M. Berridge, M. R. Huber, and A. Bergner, T. K. Flaherty, and S. Belden, T., Pozzan, P., Ferdek, C., Thorne, V., J. Gerasimenko, K., H. Baumgartner, M. Barbacid, A., Enyedi, L., Hegedus, K., Paszty, and A., Apati, J. P., E. J. Brouland, Zoelen, A., van Arbabian, P., W. Meerwijk, van H., P. Peters, H., W. Almirza, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.141408/-/DC1 online available material Supplementary material Supplementary uny .D,Hri,R,Gnails .L,Jsps .B,Re .M,Sorli, M., S. Roe, B., M. Josephs, L., N. Gandarillas, R., Harris, D., T. Bunney, rea,C,Mle,R . mt,I,Bi . Molgo T., Bui, I., Smith, A., R. Miller, C., ´rdenas, 1616-1627. Tae3epeso sa al vn uigtemlitppoeso colon of process multistep the during event early an is carcinogenesis. expression 3 ATPase B. r opooial n ucinlyhtrgnoswti cells. within heterogeneous functionally and morphologically are 15.Brigo,M:Aaei Press. Academic MA: Burlington, 51-56. signalling. calcium of universality signal. a2)utk eed ntesailadtmoa rfl fctslcCa(2+) cytosolic of profile temporal and spatial the signals. on depends uptake al. Ca(2+) et A. 1,4,5-trisphosphate. G. inositol to Mignery, Biol. response B., Cell in to ER A. J. receptors the Holmes, 1,4,5-trisphosphate from release inositol J., calcium with regulate S. interacts Conway, functionally J., Bcl-2 M. (2004). Berridge, D., M. stores. internal from Chem. release Ca2+ 1,4,5-trisphosphate-induced inositol M. A. Hofer, and mitochondria. to al. transfer et Ca2+ I. receptor 142 InsP3 Parker, constitutive J., by Yang, bioenergetics H., K. Cheung, .D. A. Biol. Cell Rev. Annu. J. FASEB A. A. remodelling. and 4 homeostasis dynamics, signalling: cancer Calcium lung cell non-small and small lines. in cell altered is Ca2+-homeostasis reticulum cancer. in Ca(2+)-store cancers. opening. (mPTP) pore Gerasimenko, transition 284 and permeability J. mitochondrial A. Watson, for R., Sutton, V. H., O. O. Petersen, V., A. Tepikin, differentiation. cell cancer lung 5408-5418. during B. expression pump Papp, calcium and C. Chomienne, and fibroblasts. oscillations NRK calcium of F(2alpha)-induced activity pacemaking prostaglandin in subtypes P. receptor A. Theuvenet, 20) tutrladmcaitcisgt norsascaindmisof domains association ras into al. insights et P. epsilon. C. mechanistic C Ponting, phospholipase and D., Esposito, F., Structural Rodrigues-Lima, F., (2006). H. Paterson, C., S. GTPases. small and messengers 517-529. , 270-283. , 20796-20803. , 20) h oso ac/nolsi eiuu acu transport calcium reticulum sarco/endoplasmic of loss The (2005). 20) ye3ioio ,,-rshsht eetrmdltscl death. cell modulates receptor 1,4,5-trisphosphate inositol 3 Type (2000). 20) nrclua acu inln.In signaling. calcium Intracellular (2003). requirement Ca2+ main the is mitochondria in elevation Calcium (2009). Nature 278 .Bo.Chem. Biol. J. 40 xetRv o.Med. Mol. Rev. Expert .Ep ln acrRes. Cancer Clin. Exp. J. 14 39503-39508. , 405-412. , 18) a genes. ras (1987). 166 1375-1379. , 395 20) acu irdmis raiainadfunction. and organization microdomains: Calcium (2006). 193-203. , m .Pathol. J. Am. ´le 645-648. , br,P,Kova P., ´bart, 20) eseseto h fet flmnl[a+ on [Ca2+] luminal of effects the of reassessment A (2003). 21) ifrn oe fioio 1,4,5-trisphosphate inositol of roles Different (2010). 7 601-632. , 276 niacr gnsMd Chem. Med. Agents Anticancer. o.Cell Mol. 26411-26420. , 21) E n A niiosfrBRAF-mutated for inhibitors RAF and MEK (2012). 20) hshlps pio:lnigsecond linking epsilon: C Phospholipase (2006). 20) euaino h nolsi reticulum endoplasmic the of Regulation (2008). 19) euaosadefcoso a proteins. ras of effectors and Regulators (1991). nu e.Biochem. Rev. Annu. 167 14 c,T,Eof . rsmn,J n Papp, and J. Grossmann, J., Enouf, T., `cs, 21) ouaino nolsi reticulum endoplasmic of Modulation (2012). a.Rv o.Cl Biol. Cell Mol. Rev. Nat. rnsCl Biol. Cell Trends 233-242. , e17. , 21 28 495-507. , 25. , elCalcium Cell 21) seta euaino cell of regulation Essential (2010). 19) acu ieaddeath and life a – Calcium (1998). adoko elSignaling Cell of Handbook 20) h estlt and versatility The (2000). 16 . Mu J., , ´ 56 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 47 640-648. , 779-827. , 544-553. , 8 20) Endoplasmic (2009). 20) Mitochondrial (2001). 705-709. , 20) Mitochondria (2002). 1 11-21. , lr . as H., Vais, M., ller, ¨ 20) Calcium (2003). .Bo.Chem. Biol. J. ESJ. FEBS MOJ. EMBO .Biol. J. pp. , 280 Cell Cell 21 , , Csorda Csorda ul,M n onad J. Downward, and M. Cully, J. P. Lockyer, and J. P. Cullen, J. Cre P. Lockyer, and J. S. Cook, rniwc,G,Pei,M n se,R Y. R. Tsien, and M. Poenie, G., Grynkiewicz, owy .J,Tuig .W,Ade,S,Kilu,B . lwlnRoderick, Llewelyn T., B. Kvinlaug, S., Andreu, W., J. Thuring, J., S. Conway, aa,R . ugthe,A . ahno,M .adErih .E. B. Ehrlich, Hajno and H. M. Nathanson, D., A. Burgstahler, E., R. Hagar, Lebiedzinska, R., M. Wieckowski, C., Santangelo, K., H. Lin, K., Ito, C., Giorgi, onna,A. Hovnanian, Ritter, M., E. Corps, A., Talasila, M., F. Drawnel, J., C. Fearnley, R., D. Higazi, rwe,F . ahe,D,Mletn .D,Milt . rne,J . Swift, M., J. Aronsen, M., Maillet, D., J. Molkentin, D., Wachten, M., F. Drawnel, J. Downward, J. Downward, asn .J,Bomn .D,Dsehrt .W,Wjiiwc,R .and J. R. Wojcikiewicz, W., C. Distelhorst, D., M. Bootman, J., Roderick, C. and T. Hanson, Maraldi, W., C. Distelhorst, D., M. Bootman, J., C. Hanson, A. R. Weinberg, and D. Hanahan, P. Pizzo, A., Gianelle, M., Scorzeto, M., Bortolozzi, I., Drago, M., Giacomello, R. M. Duchen, egwy . etce,D . oa,P . lemt,H .adCwn R. Cowen, and J. H. Altermatt, R., P. Hoban, C., D. Betticher, J., T., Heighway, Nakamura, T., Michikawa, H., Miyauchi, T., Higo, Z., A. Suzuki, M., Hattori, H. Higashida, and Y. Nozawa, M., Hashii, Conway, A., Tashfeen, O., Ritter, S.-Y., R. Foo, M., Movassagh, D., Harzheim, omtc,R . u .adLws .S. R. Lewis, and K. Xu, E., R. Dolmetsch, Szabo S., Campello, U., Marchi, De aaa,D n eneg .A. R. Weinberg, and D. Hanahan, etrsadsgiiac ftepyia ikg ewe Radmitochondria. and ER between Biol. linkage Cell physical J. the of significance and features a.Rv o.Cl Biol. Cell Mol. Rev. Nat. interface. ER-mitochondrial the at 132. dynamics Hajno calcium local and T. Balla, Hajno and A. C. 2b Mannella, SERCA T., to due content reticulum endoplasmic over-expression. increasing by C. proliferation Slomianny, and N. Prevarskaya, proliferation. cell and regulation RAS 887-893. ematdrvtvso y-nstl1,4,5-trisphosphate. myo-inositol B. A. of Holmes, derivatives and D. permeant M. Bootman, L., H. yeIIIs3rcpo hne ty pni h rsneo increased of presence the in open stays channel receptor calcium. InsP3 III Type properties. fluorescence improved 3440-3450. greatly with indicators release. calcium al. modulating et by R. reticulum Bernardi, endoplasmic J., Science at Duszynski, M., apoptosis Bonora, regulates A., Bononi, M., 45 myocytes. hypertrophic cardiac L. for in nexus H. a Roderick, signaling is and release D. Ca2+ M. InsP3-induced Bootman, Endothelin-1-stimulated K., Mikoshiba, F., McDonald, O., . jatd . i,N,Ctlci . iohb,K tal. et K. Mikoshiba, D., Catalucci, N., Liu, I., Sjaastad, F., Cancer Rev. pathways? parallel or transactivation ras: 376 of regulation receptor oeik .L. H. Roderick, effect. apoptotic L. H. transport. calcium mitochondrial of store- of activation by not but stores, from channels. mobilization Ca2+ operated Ca2+ by generated T. Pozzan, and Physiol. J. and signals calcium regulates miRNA-133a hypertrophy. and cardiac IP(3)RII between antagonism 19) omlfcto ntmr fKA2 ye2ioio ,, triphosphate KRAG. 1,4,5 gene, inositol human 2 novel type a KRAS2, and gene, of receptor tumors in Coamplification signaling. (1996). Ca2+ in 3 and 1 types receptor K. Mikoshiba, and T. Inoue, voltage- in fibroblasts. influx NIH/3T3 Ca2+ ras-transformed clamped tetrakisphosphate-induced inositol and oscillations Ca2+ arrhythmias and transients hypertrophy. 11411. Ca2+ cardiac L. with augment H. associated Roderick, reticulum and sarcoplasmic D. junctional M. Bootman, J., S. h fiinyadseiiiyo eeexpression. gene of specificity and efficiency the mitochondria. isolated of transition M. e1. 1292 generation. 70. ,,-rshsht eetr n niisC(+ paeb mitochondria by uptake content. Ca(2+) store calcium inhibits ER affecting and without receptors 1,4,5-trisphosphate pn . iax . adnAel,F,Dwil,E,Gfi,V., Goffin, E., Dewailly, F., Vanden-Abeele, G., Bidaux, A., ´pin, 337-363. , zy .adCsorda and G. czky, ´ 20) a osntdrcl atcpt nteC(+-nue permeability Ca(2+)-induced the in participate directly not does Bax (2004). e9-e10. , s . Va G., ´s, Va C., Renken, G., ´s, 20a.Teclua ocnrto fBl2dtrie t r-o anti- or pro- its determines Bcl-2 of concentration cellular The (2008a). ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal 330 Nature 529 Cell 174 1247-1251. , 20b.Treigrssgaln ahasi acrtherapy. cancer in pathways signalling ras Targeting (2003b). 20) iohnraadclim rmcl inligt eldeath. cell to signalling cell from calcium: and Mitochondria (2000). 3 20a.Rl frcpo yoieknssi G-protein-coupled in kinases tyrosine receptor of Role (2003a). 20) EC up n ua diseases. human and pumps SERCA (2007). ra,P,Golena P., ´rnai, 11-22. , 57-68. , 915-921. , 396 144 elCalcium Cell ice.J. Biochem. 21) a+htsoso h iohnra ufc are surface mitochondrial the on spots hot Ca2+ (2010). 20b.Bl2sprse a2)rlaetruhinositol through release Ca(2+) suppresses Bcl-2 (2008b). 81-84. , 646-674. , .Cl Biol. Cell J. zy G. czky, ´ s G.´s, 3 ra,P,Wle,L,Wae,D,Btl,K . Balla, F., K. Buttle, D., Weaver, L., Walter, P., ´rnai, o.Cell Mol. 339-348. , 20) itntrlso nstl1,4,5-trisphosphate inositol of roles Distinct (2004). 20) npht a signaling. ras SnapShot: (2008). 401 44 20) nerto fclimadrssignalling. ras and calcium of Integration (2002). r . o,S,Pris . cnie,T G., T. Schneider, G., Purkins, S., Roy, T., ´r, 21) acu inlig ihn u molecules out fishing signalling: Calcium (2010). o.Cell Mol. 20) eetavne nCa(2+)-dependent in advances Recent (2006). 21) mgn neognlecnat and contacts interorganelle Imaging (2010). 243-258. , . obl,F,Mrio,J .adZoratti, and C. J. Martinou, F., Tombola, I., , ` zy G. czky, ´ 20) h alak fcancer. of hallmarks The (2000). 199 49-55. , .Bo.Chem. Biol. J. ur Biol. Curr. 38 783-798. , elCalcium Cell rc al cd c.USA Sci. Acad. Natl. Proc. 21) alak fcne:tenext the cancer: of Hallmarks (2011). 20) rlci tmltspott cell prostate stimulates Prolactin (2007). 280-290. , 33 19) acu siltosincrease oscillations Calcium (1998). 19) rdknnidcdcytosolic Bradykinin-induced (1993). .Bo.Chem. Biol. J. 472-482. , 20) h ytei fmembrane of synthesis The (2006). elCalcium Cell 20) tutrladfunctional and Structural (2006). 18) e eeaino Ca2+ of generation new A (1985). 20) nrae nPR nthe in InsP3Rs Increased (2009). .Bo.Chem. Biol. J. 20 R888-R891. , Nature Genomics 279 39 101-112. , 37415-37422. , 392 44 279 .Bo.Chem. Biol. J. ut .Chem. J. Aust. 324-338. , 268 o.Cell Mol. ucl.Biochem. Subcell. 35 933-936. , 11967-11975. , Cell 207-214. , 19403-19410. , 21) Mutual (2012). Cell 21) PML (2010). ice.J. Biochem. 106 133 39 11406- , 100 1292- , (1998). (2009). 121- , 57- , 260 Nat. 59 , ,

Journal of Cell Science aci . aiel,M,Bnn,A,Bnr,M,Gog,C,Rmsi .and A. Rimessi, C., Giorgi, M., J. Bonora, S. A., Bononi, Cook, M., Marinello, and S., D. Marchi, P. Smith, S., A. Little, Lompre and S. J. F., Hulot, Abeele, L., Lipskaia, Vanden E., Dewailly, C., Slomianny, S., Humez, G., Legrand, Wo E., Kahn, F., Friedrich, F., Lang, ite,R,Pt,G,Pleir . oe,I,Hnne,A . rcsl D., Drechsel, K., A. Koros Heninger, I., Poser, L., K. Pelletier, S. Joseph, G., and Putz, C. R., Bhanumathy, I., Kittler, D. Yule, I, I. L., Wagner, T., M. Khan, itn . err,D,Rpzi . iVrii,F,Pza,T n izt,R. Rizzuto, and T. Pozzan, F., Virgilio, Di E., Rapizzi, D., Ferrari, P., Pinton, B., A. Holmes, J., S. Conway, L., Mackenzie, J., T. Collins, M., Y. C. R. Peppiatt, Tsien, and C. J. Reed, C., Jin, E., A. Palmer, M., P. Nicotera, Nishino, and C., B. Zhivotovsky, M. S., Orrenius, Bassik, T., J. Opferman, L., Scorrano, A., S. Oakes, Hyzinski-Garcı J. K., R. S. Motiani, Roberts-Thomson, and M. F. J. Davis, S. R., Roberts-Thomson, G. and Monteith, M. H. Faddy, D., McAndrew, R., G. Monteith, ely .G,Rk,S . nrk,J .adSrk,A V. A. Smrcka, H., S. and Li, M., D. M. Sabatini, G., J. Schilling, H., A. Ondrako, Sharp, J., M. E., Soloski, A., S. A. Khan, Reks, G., G. Kelley, He L., Verbert, L., S. Kocks, N., N. Kasri, iaaa . ad,A,Ymzw,T,Hrs,K,Krsk,T n io M. Iino, and T. Kurosaki, K., Hirose, T., Yamazawa, A., Maeda, T., Miyakawa, ar,N . oms .M,Blyc,G,Prs .B,Bomn .D., M. Bootman, B., J. Parys, G., Bultynck, M., A. Holmes, N., N. R. Kasri, A. Means, and R. C. Kahl, Lepage, P., Marchetti, M., Monet, F., Vanden-Abeele, G., Legrand, S., Humez, ARTICLE RESEARCH eds .C,Gms .A,Topo,M,Suo .C,Ge,T . Goes, S., T. Goes, C., N. Souto, M., Thompson, A., D. Gomes, C., C. Mendes, itn P. Pinton, cell inhibitors. of pathway control ERK1/2 the to resistance in activity ATPase calcium proliferation. and and growth content in calcium involvement reticulum 2B control. Ca2+-ATPases growth cell sarcoendoplasmic cancer N. prostate for human Prevarskaya, and Evidence F. growth. Wuytack, P., Mariot, oncogene. Ha-ras the expressing 4942. cells in potential H. cancer. Grunicke, and lung K. and colon with correlation in Cytogenet. gene ATP2A2 the noioulaepeae iN ceni ua el dniisgenes identifies cells human division. in cell al. for et screen essential H. Grabner, siRNA B., Habermann, endoribonuclease-prepared I., Konstantinova, S., receptors. Fischer, 1,4,5-trisphosphate inositol of Chem. phosphorylation Biol. J. kinase Akt (2006). 20) h a+cnetaino h nolsi eiuu sakey a is molecular reticulum the for endoplasmic significance the action. apoptosis: Bcl-2 of of ceramide-induced mechanism concentration of Ca2+ determinant The (2001). channels. and entry use-dependent calcium L. a 34 store-operated has H. on and action Roderick, pumps reversible and calcium slowly T. inhibits 1,4,5-trisphosphate- release, J. inositol calcium Seo, induced antagonises J., (2-APB) M. borate Berridge, Aminoethoxydiphenyl improved D., M. an Bootman, with analyzed sensor. Ca2+ fluorescent 17404-17409. reticulum encoded endoplasmic genetically in alterations link. calcium-apoptosis the endoplasmic the human from leak calcium for and receptor reticulum. trisphosphate essential inositol 1 J. type are S. the Korsmeyer, and and T. Pozzan, activity channel M. Trebak, CRAC and A. invasion. mediate A. glioblastoma Mongin, Orai1 K., Matrougui, and H., Y. Kuo, F., I. consequences. and changes cancer: in 287 pumps and channels transport. Ca2+ 530. targeting cancer: and Calcium (2007). nrae ye3ioio ,,-rshsht receptor. 1,4,5-trisphosphate H. inositol S. 3 Snyder, type and increased A. C. Ross, effector. ras novel a C(epsilon): Phospholipase H. Smedt, cells. knock-out endoplasmic- double 51. decreased De presenilin a in content for and responsible Ca2+ reticulum is L. 1 type Missiaen, receptor 1,4,5-trisphosphate B., J. 19) noigo a+sgasb ifrnilepeso fI3receptor IP3 of expression differential by signals Ca2+ subtypes. of Encoding (1999). Ca itof . isan . coad . eSet . owy .J tal. et J. proteins. Ca2+-binding S. neuronal Conway, by J. H., activity EMBO Smedt, receptor InsP3 De F., of Regulation McDonald, (2004). L., Missiaen, K., Rietdorf, regulation growth TNFalpha. cell and cancer IGF prostate N. by in Prevarskaya, content and calcium F. reticulum Wuytack, endoplasmic E., Dewailly, A., Crepin, G., .M,Rdius .A,Gmz .V,Ntasn .H n et,M F. M. Leite, and H. transmits mitochondria. M. preferentially into Nathanson, receptor signals V., 1,4,5-trisphosphate Ca2+ M. inositol apoptotic III Gomez, type A., The M. (2005). Rodrigues, M., A. acu/amdlndpnetpathways. calcium/calmodulin-dependent 97-108. , 2+ c . lvc . ot .adRvi-lvc M. Ravnik-Glavac, and T. Rott, D., Glavac, B., ec, ˇ 31666-31673. , ees n apoptosis. and release 23 MOJ. EMBO rc al cd c.USA Sci. Acad. Natl. Proc. 21) eetv ouaino utp I IP III subtype of modulation Selective (2012). 171 312-321. , 281 105-111. , 3731-3737. , 18 19) rdknnidcdoclain fcl membrane cell of oscillations Bradykinin-induced (1991). 1303-1308. , fuesArch. Pflugers .Cl.Physiol. Cell. J. fuesArch. Pflugers Nature a.Rv o.Cl Biol. Cell Mol. Rev. Nat. MOJ. EMBO a .C,Zag . ekl .M,Abdullaev, M., M. Henkel, X., Zhang, C., M. ´a, elDahDis. Death Cell 20) euaino elcceporsinby progression cycle cell of Regulation (2003). 432 19) ypoyeaotss eito by mediation apoptosis: Lymphocyte (1996). l . amrr . adge,S,Maly, S., Waldegger, M., Hammerer, E., ll, ¨ 20) raottcBXadBKregulate BAK and BAX Proapoptotic (2005). ,A M. A. ´, 1036-1040. , 20 465 102 201 457 2690-2701. , Oncogene 1249-1260. , br,S . alwet . Parys, G., Callewaert, S., S. ´bert, nor Rev. Endocr. 105-110. , .Bo.Chem. Biol. J. rc al cd c.USA Sci. Acad. Natl. Proc. 201-213. , .Bo.Chem. Biol. J. 673-685. , 20) oeo sarco/endoplasmic of Role (2009). 20) prglto finositol of Up-regulation (2006). 3 21) ehnsso acquired of Mechanisms (2013). e304. , 20) euaino eldeath: cell of Regulation (2003). MOJ. EMBO 20) a+posadcell and pools Ca2+ (2001). .Bo.Chem. Biol. J. 4 32 552-565. , a.Rv Cancer Rev. Nat. Science 1207-1215. , 20) Bcl-2-mediated (2004). 3 24 yAtrgltsER regulates Akt by R 280 20 20) leain in Alterations (2006). 719-736. , 276 elCalcium Cell 743-754. , 40892-40900. , 47608-47614. , 21) Calcium (2012). 273 acrGenet. Cancer 20) oeof Role (2004). .Bo.Chem. Biol. J. 21) STIM1 (2013). elCalcium Cell 503-507. , 266 20) An (2004). 20) 2- (2003). 4938- , (2001). 7 40 519- , ,41- 101 , izt,R,Pno,P,Crigo,W,Fy .S,Fgry .E,Lfht,L M., L. Lifshitz, E., K. Fogarty, S., F. Fay, W., Carrington, P., Pinton, R., Rizzuto, R. Rizzuto, and E. Zecchini, R., Siviero, C., Giorgi, P., Pinton, cuz,A,Nce . an,P . olno,S n onad J. Downward, and S. Tomlinson, H., P. Warne, B., Nicke, A., Schulze, S., Kin, T., Takagi, K., Shimomura, K., Fukuda, A., Hagiwara, C., Sakakura, J. S. Cook, and L. H. Roderick, Rizzuto,R.,DeStefani,D.,Raffaello,A.andMammucari,C. izt,R n ozn T. Pozzan, and R. Rizzuto, W. B. Warner, R., C. Erwin, H., L. Liu, L., M. Miller, P., G. Boivin, V., Prasad, ohd,J,Iauh,K,Mtu,T,Ihbsi . auk,T n iho M. Nishio, and T. Masuoka, T., Ishibashi, T., Matsui, K., Iwabuchi, Bobe, J., Yoshida, L., Dode, L., Raeymaekers, H., Verboomen, B., Papp, F., Wuytack, R. Casteels, and L. Plessers, L., Raeymaekers, A., J. Eggermont, F., Wuytack, K., F. Chan, F., K. To, S., S. Ng, X., M. Luo, S., A. Cheng, J., X. Wang, K., W. Wu, and J. C. G. Marshall, Weber, I., McKay, D., M. Houslay, A., S. Davies, J., M. T., Wakelam, Sasazuki, S., Shirasawa, L., Li, J., M. Brauer, C., Bentley, S., Vartanian, oeik .L,Cmbl,A .adLeeln .H. D. Llewellyn, and K. A. Campbell, L., H. Roderick, T. Pozzan, and R. M. Duchen, R., Rizzuto, Magalha G., Szabadkai, M., Chami, D., Ferrari, P., Pinton, R., Rizzuto, ueo,D . hw .T,Wli,N . ivr .P,Jcsn .L,Chang, L., E. Jackson, P., D. Silver, and A., Y. N. R. Willis, T., Tsien, A. W., Shaw, Li, A., D. J., Tuveson, M. Berridge, C., S. Tovey, P., Lipp, D., Thomas, W. S. Lowe, and D. Beach, E., M. McCurrach, W., A. Lin, M., Serrano, ztosi . ay,J . uddAioc,H n aia,F. Matifat, and H. Ouadid-Ahidouch, B., J. K., Parys, Rietdorf, C., Szatkowski, H., Smedt, De B., J. Parys, V., Vanderheyden, T., Szado, R. M. Duchen, and T. G. Sasazuki, Szabadkai, and N. Yokoyama, M., Furuse, S., Shirasawa, eiuu sdtriat fmtcodilC2 responses. Ca2+ mitochondrial T. of determinants Pozzan, as and reticulum A. R. Tuft, novmn fioio ,,-rshsht eetrtp I33 nthe in (IP3R3) 3 type receptor cancers. al. gastric 1,4,5-trisphosphate et of Y. dissemination inositol peritoneal Okazaki, K., of Mikoshiba, involvement J., Fujiyama, Y., Nakase, signalling. calcium of regulators Biol. and Cell sensors as Mitochondria eemnnsadfntoa consequences. functional and determinants susceptibility. cancer of mode novel a via Res. tumors cell squamous predisposes to pump, Ca2+ mice 2 isoform apoptosis. Ca2+-ATPase reticulum sarco(endo)plasmic of control andShull,G.E. the in transfer Ca2+ Oncogene ER-mitochondria apoptosis: and prtdclimety elpoieainadtmrgnct nhuman in tumorigenicity and proliferation cells. cell A431 carcinoma entry, store- epidermoid suppresses calcium (STIM1) 1 molecule operated interaction stromal of Knockdown (2012). cells. mast in expressed and is cells, lymphoid pump in Ca2+ platelets, in 3-type Ca(2+)-ATPase reticulum sarco/endoplasmic R.,Enouf,J.,Bokkala,S.,Authi,K.S.andCasteels,R. reticulum endoplasmic the of ATPase. isoform Ca2(+)-transport non-muscle the against Antibodies (1989). J. Yu, and carcinogenesis. J. colon 86 in J. pathways Sung, signaling cellular H., C. Cho, production. phosphate inositol to receptors lines. cell A. isogenic Hall, of panel a al. Chem. using et Biol. phenotypes Z. J. K-Ras-dependent Modrusan, E., mutant Stawiski, P., of Haverty, S., J. Kim, h rncitoa epnet a ciaini lotcmltl dependent completely almost is activation Raf to response transcriptional The with interaction its by enhanced is vivo in receptors. calreticulin glucocorticoid of localisation survival. and proliferation cell 361-375. cancer for Ca2+ remodelling liaison. Ca2+ mitochondria T. Pozzan, and F. hypotheses. Virgilio, Di 1763-1766. . ecr .L,Gohw . ok . rwe,D tal. et D. Crowley, H., Hock, widespread and R., defects. proliferation developmental Grochow, and stimulates neoplastic K-ras(G12D) L., oncogenic K. Endogenous Mercer, S., cells. non-excitable in sites D. M. Bootman, autocrine major a shows and activity component. kinase kinase protein mitogen-activated on ua acrcells. cancer human nstl145tipopaeidcdC2 inligi novdi estradiol- in growth. involved cell epithelial is cancer signalling breast induced Ca2+ 1,4,5-trisphosphate-induced Inositol protein by apoptosis. and receptors release 1,4,5-trisphosphate 105 Ca2+ So inhibits inositol U., B/Akt Landegren, of kinase F., Phosphorylation Khan, E., (2008). Chastre, L., Kotelevets, Ki-ras. signaling. activated at disrupted lines cell cancer 260 colon human of growth p16INK4a. and p53 accumulationof with associated senescence cell premature provokes ras Oncogenic 251-277. , 2427-2432. , 85-88. , 65 ora fCl cec 21)17 6711 doi:10.1242/jcs.141408 1607–1619 127, (2014) Science Cell of Journal 8655-8661. , 18) omlp1-a ope obsnadohrgot factor growth other and bombesin couples p21N-ras Normal (1986). 20) onrglto ficesdsga rndcincpct in capacity transduction signal increased of Down-regulation (2005). 13 hsooy(Bethesda) Physiology 27 566-578. , o.Bo.Cell Biol. Mol. Oncogene 6407-6418. , 288 20) irsoi rpriso lmnayC2 release Ca2+ elementary of properties Microscopic (2000). 2403-2413. , d.Ezm Regul. Enzyme Adv. 20) alisfiinyo t22 noigthe encoding Atp2a2, of Haploinsufficiency (2005). Cell 22 ESLett. FEBS 20) irdmiso nrclua a+ molecular Ca2+: intracellular of Microdomains (2006). ice.J. Biochem. 8619-8627. , 88 15 c.STKE Sci. ur Biol. Curr. 593-602. , 3450-3463. , 19) ls otcswt h endoplasmic the with contacts Close (1998). 20) a+sgaln hcpit ncancer: in checkpoints signalling Ca2+ (2008). 20) iohnra h u fclua Ca2+ cellular of hub the Mitochondria: (2008). 23 20) acu n ppoi:fcsand facts apoptosis: and Calcium (2003). ice.Pharmacol. Biochem. 84-94. , 405 2004 10 264 21) yrglto n rstl of crosstalk and Dysregulation (2013). acrCell Cancer 181-185. , 8-15. , 20) lrigi itesae h ER/ the space: little in Flirting (2004). 45 765-769. , Nature niacrRes. Anticancer re1. , hso.Rev. Physiol. o.Cancer Mol. 37-51. , .Bo.Chem. Biol. J. 323 rc al cd c.USA Sci. Acad. Natl. Proc. 5 rt e.Ocl Hematol. Oncol. Rev. Crit. 375-387. , 173-176. , 84 9 21) Identification (2013). 86 a.Rv Cancer Rev. Nat. ebr,O tal. et O. derberg, ¨ 156. , 1592-1603. , 35A 23 369-408. , 20) Possible (2003). 269 19) Nuclear (1997). 20) Calcium (2008). 19) Altered (1993). a.Rv Mol. Rev. Nat. Science 1410-1416. , 3691-3697. , 19) A (1994). e,P J., P. ˜es, Science (2012). Cancer (2004). (2010). (2004). (1997). 1619 280 8 , ,

Journal of Cell Science