PARP1-Mediated Necrosis Is Dependent on Parallel JNK and Ca

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erss u o api ciain ncnrs,ihbto fcalpain of inhibition Ca contrast, by In either activation. calpain not but necrosis, MNNG- reduced significantly inhibition and JNK translocation. activation AIF JNK activity, and calpain in increases with associated was This or (MNNG) fMsor-ouba ouba isui 51,USA. 65211, Missouri, Columbia, Missouri-Columbia, of ocuin lhuhJKadclanbt otiuet PARP1- mechanisms. to parallel via contribute so both do they calpain necrosis, induced and JNK and although conclusion, MNNG- abrogate to pore MPT failed the and Bax AIF, RIP1, genetic of surprise, inhibition pharmacological our and/or To translocation. AIF or activation JNK affect not Ato o orsodne([email protected]) correspondence for *Author Columbia, USA. Missouri-Columbia, 65211, of Missouri, University Physiology, and Pharmacology ouba isui 51,USA. 65211, Missouri, Columbia, in .Douglas L. Diana and JNK parallel on Ca dependent is necrosis PARP1-mediated ARTICLE RESEARCH ß 4134 2014 July 8 Accepted 2014; January 25 Received 1 under kinases the serine/threonine of receptors the formation of the (TNF) consisting to complex factor leads a inhibition necrosome, necrosis necroptosis. caspase-8 of or tumor conditions necrosis, death-receptor- of on programmed focused Activation of have a mechanisms also studies particular, recent In induced pathways of 2012). molecular al., number recent et great distinct However, (Vanlangenakker cell. that necrosis the determined mediate indicated of process death has uncontrolled genetically ‘accidental’ random, evidence the it a specific, to recently was leads until are necrosis that contrast, that In there believed process. was this that due mediate death that fact cell pathways of form the ‘programmed’ a to as regarded is Apoptosis INTRODUCTION AIF, Calpain, JNK, PARP1, Mitochondria Necrosis, death, Cell WORDS: KEY fibroblasts embryonic Mouse the tested. of either be with treated PARP1-induced components to were (MEFs) different yet simply for has two pathway are same they pathways these whether or distinct whether necrosis, However, of represent release (AIF). mechanisms mitochondrial factor and Bax apoptosis-inducing of involving kinases other activation JNK the and calpain-mediated (MPT), RIP1 transition involving permeability one mitochondrial proposed: and been mechanisms have main this Two for necrosis. that caspase-independent enzyme trigger nuclear can a is (PARP1) polymerase-1 Poly(ADP-ribose) ABSTRACT atnCrivsua eerhCne,Uiest fMissouri-Columbia, of University Center, Research Cardiovascular Dalton 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,43–15doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. b 2+ LpcoeidcdJKatvto,AFtasoain and translocation, AIF activation, JNK -Lapachone-induced clanpathways /calpain b 2+ Lpcoe eutn nPR1dpnetnecrosis. PARP1-dependent in resulting -Lapachone, hlto rkokonatnae erss u did but necrosis, attenuated knockdown or chelation 1 n hitpe .Baines P. Christopher and 2 eateto imdclSine,University Sciences, Biomedical of Department N -methyl- b Lpcoeidcdncoi.In necrosis. -Lapachone-induced N 9 -nitro- 3 eateto Medical of Department N -nitrosoguanidine 1,2,3, * nue erssi eedn natvto fteCa PARP1- the that of activation reported on have dependent is 2007) and necrosis al., Moubarak induced defined. et clearly be (Moubarak to colleagues yet have death cell mediated 2006; Chiu 2011). 2010; al., al., al., et et et Park Dong 2010; 2011; (Xu al., al., et necrosis et pathways Artus 2007; PARP1-induced 2005). molecular al., et that Hageman, Moubarak specific appreciated and more now Wijk follows is (van it ATP However, subsequently and cell, uha xdnsadakltn gns aeln been long have agents, alkylating and stresses, oxidants Genotoxic emerged. as poly(ADP-ribose) also enzyme such has repair (PARP1) DNA polymerase-1 the involving program 2012). al., like et Wang kinase 2012; al., lineage et (Sun mixed PGAM5 phosphatase pseudokinase mitochondrial-associated the of the activation subsequent 2011) with (MLKL) al., of et Vanlangenakker reactive activation mitochondrial 2011; and al., of et induction (Irrinki 2007), species This killingoxygen al., NADPH 2012). necrotic et of al., facilitate activation (Kim including et to oxidases mechanisms, Li proposed of 2009; variety been a al., al., has through et et turn Zhang (Cho in 2009; 3 complex al., and et 1 RIPK) He or 2009; (RIP, proteins interacting receptor hr h vrciePR1ue ptespl fNAD of supply the up used PARP1 catastrophe, overactive by metabolic the induced to necrosis where due that simply thought was originally hyperactivation was PARP1 It 1998; 2010). al., al., et et (Zingarelli an Li PARP1 of with inhibition genetic ischemia/ associated markedly by is attenuated Moreover, is necrosis cerebral 2009). that and myocardial (Hassa, death reperfusion-induced PARP1 cell of necrotic overstimulation cause to known nue ytodsic AP activators, PARP1 necrosis distinct PARP1-mediated in two MPT by and Bax induced calpain, JNK, RIP1, of role the investigate systematically PARP1 activator pharmacological distinct single another lacking. with a are comparisons only of direct majority of on and the activator, components Moreover, two relied tested. different be have to simply studies yet these PARP1-induced has are pathway for they same the whether whether pathways or distinct necrosis, However, represent mechanisms cyclosporine-A. pore be (MPT) activates can transition that inhibitor permeability turn activation mechanism mitochondrial nuclear in the PARP1 a by through that RIP1 blocked necrosis and suggested RIP1. induces have which kinase release JNK1, 2006) pro-necrotic al., the the et Xu activates contrast, (Xu In elicits al. (AIF). et factor the it apoptosis-inducing to of translocation translocation Bax where induces turn mitochondrion in which calpain, protease I1 a,AF n P pert edispensable. be to appear MPT necrosis, and PARP1 AIF, in calpain Bax, and RIP1, JNK for roles independent confirm to and (MNNG) nitrosoguanidine ept hs h inln ewrsrsosbefrPARP1- for responsible networks signaling the this, Despite nadto otencotssptwy nte necrotic another pathway, necroptosis the to addition In osqetyteproeo h rsn td a to was study present the of purpose the Consequently b Lpcoe lhuhw eeable were we Although -Lapachone. n ucinlinterrelationships functional and s N -methyl- 2+ N -activated 9 -nitro- + nthe in N -

Journal of Cell Science motnl,tecttxcefc fbt opud was compounds both 1A). of (Fig. effect necrosis cellular cytotoxic substantial the wild-type both induced Importantly, of with MNNG Treatment (MEFs) and PARP1. fibroblasts of embryonic mouse activation through that acting verified first We necrosis independent b RESULTS ARTICLE RESEARCH tiig nwl-ye( wild-type in staining, necrosis. independent, 1. Fig. small RIP1-specific a with PARP1-mediated pro- MEFs in the JNK of of role and Transfection the RIP1 examined necrosis. next kinases al., we et signaling 2011), (Xu necrotic al., proposed et been Chiu has 2006; pathway PARP1–RIP1–JNK a As RIP1 not but JNK b MEFs. in apoptosis, than rather necrosis, PARP1-dependent both that confirmed data These benzyloxycarbonyl- 1C). inhibitor inducer (Fig. pan-caspase (zVAD-FMK) Val-Ala-Asp(OMe)-fluoromethylketone the apoptosis to canonical insensitive was to and a contrast 1B), in no staurosporine, (Fig. cells observed treated with the We in treatment 1A). cleavage (Fig. caspase-3 of MEFs evidence PARP1-deficient in ablated essvehicle; versus 20 with treated xeiet efre ndpiae eut r mean are zVAD-FMK Results inhibitor duplicate. pan-caspase in the performed without experiments or with h 4 (20 for MNNG mM 1 or 20 with treated tuoprn o 8hwsue sapstv oto.()Ncoi,as Necrosis, 20 (C) with treated control. MEFs positive in a staining, Green as Sytox nM used by 300 was measured with h Treatment 18 GAPDH. for and staurosporine caspase-3 cleaved for blotted Lpcoe n NGidcdncoi sdpneton dependent is necrosis MNNG-induced and -Lapachone- caspase- PARP1-dependent, induce MNNG and -Lapachone m ) eut hw r ersnaieo he rfu independent four or three of representative are shown Results M). b LpcoeadMN nuePR1dpnet u caspase- but PARP1-dependent, induce MNNG and -Lapachone m m À P M M , b b b .5versus 0.05 Lpcoe n NGidcdcl death cell MNNG-induced and -Lapachone- lpcoeo MMN o .()MF were MEFs (B) h. 4 for MNNG mM 1 or -lapachone lpcoeo MMN o ,adte western then and h, 4 for MNNG mM 1 or -lapachone Parp1 A erss smaue yStxGreen Sytox by measured as Necrosis, (A) +/+ b LpcoeadMN eeindeed were MNNG and -Lapachone n AP-eiin ( PARP1-deficient and ) Parp1 b LpcoeadMN induced MNNG and -Lapachone +/+ . Parp1 6 ...* s.e.m. m 2 b M / 2 -Lapachone b MEFs ) P -lapachone , 0.05 07 oge l,21) ossetwt hs otetetwith co-treatment this, with Consistent Ca 2010). the al., et al., in et Dong Moubarak 2007; signals 2003; al., proximal et (Tagliarino key necrosis MNNG-induced as calpain protease aeilFg 1,) erpae hs xeiet using experiments these repeated We S1A,B). Ripk1 Fig. Pharmacological attenuate material 2B,C). to (Fig. failed similarly agent necrostatin-1 b either with manner the RIP1 attenuate of of dose-dependent to inhibition effect failed a however, death RIP1, in cell of death Depletion by 2B,C). necrotic (Fig. levels cells induced siRNA-transfected protein control MNNG RIP1 In 2A). reduced (Fig. (si)RNA interfering ersswt nyasalrdcinosre ttehighest the at observed reduction small a only of with silencing Interestingly, either necrosis 3D). either affect (Fig. greatly down not MEFs knocking did the JNK1 by in involved JNK2 is or then JNK1 isoform We necrosis. JNK PARP1-mediated in which step tested activation crucial JNK a RIP1, be unlike to Thus, appears 3B,C). (Fig. inhibit death significantly an cell induced to SP600125, able also by SP600125 with was Co-incubation attenuated 3A). (Fig. significantly signaling JNK of inhibitor was this activation. of Moreover, indicative MEFs 3A), (Fig. of phosphorylation JNK in Treatment increase JNK. either of activation with elicited activation the PARP1 affect not did RIP1 of absence of ability complete the even However, ocnrto fteaet Fg EF.I otatJNK2 contrast causative a death. plays necrotic In that PARP1-mediated isoform in this role 3E,F). is both (Fig. it to that response suggesting in compounds agents death cell attenuated the considerably knockdown of concentration Esnl o ohBxadBk(lokona A1 to BAK1) as known (also Bak and and Bax MEFs that both wild-type as exposed for much as null then not MEFs We although staurosporine. staining, induced with Bax agents seen active Both the 5A). in (Fig. for increase protein an staining the necrosis, immunofluorescence of by therefore conformation MNNG-induced activation open We Bax in 2007). of al., level role et with key MEFs (Moubarak treated calpain a been of play has downstream Bax to protein mitochondrial Bcl-family distal postulated the the of investigated Activation next mechanisms. we necrosis mediated aigetbihdJKadCa and JNK established Having Bax on b the implicated have studies previous Ca JNK, of to mobilization addition In Ca b ereo ertccl et nrsos to response in death cell necrotic of degree eiin el Fg B tl xiie outdose-dependent robust a exhibited to Bax/Bak- still response the death surprise 4B) (Fig. our cells to deficient However, MNNG. and Lapachone ciiyo ohioom.Ti iN eue an eesto levels the Capn4 for reduced reduced required siRNA This subunit isoforms. , small both the of Capn4, activity against siRNA with the inhibit dose-dependently genetically both also was by activity increased Calpain 4A,B). (Fig. MNNG Lpcoe n NGidcdncoi sntdependent not is necrosis MNNG-induced and -Lapachone- on dependent is necrosis MNNG-induced and -Lapachone- Lpcoe n NGidcdncoi et (supplementary death necrotic MNNG-induced and -Lapachone- 5 hto oto rnfce el Fg E n markedly and 4E) (Fig. cells transfected control of that 35% ihrgrst N inln,w is xmndwhether examined first we signaling, JNK to regards With 2+ n calpain and 2 2+ / ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal 2 b hltn gn AT-Msgiiatyatnae the attenuated significantly BAPTA-AM agent chelating b Lpcoe n NGidcdncoi Fg 4F,G). (Fig. necrosis MNNG-induced and -Lapachone- Eswihaecmltl eodo I1(i.2D). (Fig. RIP1 of devoid completely are which MEFs LpcoeadMN oeoencoi Fg 2E,F). (Fig. necrosis evoke to MNNG and -Lapachone b Lpcoeo NGcue dose-dependent a caused MNNG or -Lapachone b b LpcoeadMN htwso similar of was that MNNG and -Lapachone b 2+ LpcoeadMN n eemndthe determined and MNNG and -Lapachone LpcoeadMN Fg CD.To 4C,D). (Fig. MNNG and -Lapachone m n -apisw rnfce h MEFs the transfected we m-calpains and - n ciaino h Ca the of activation and 2+ b clana eesr o PARP1- for necessary as /calpain Lpcoe rMNNG-induced or -Lapachone- b Lpcoe n MNNG- and -Lapachone- b b b Lpcoe and -Lapachone- Lpcoeand -Lapachone Lpcoeand -Lapachone 2+ -dependent , 4135 80% b -

Journal of Cell Science Escmltl eiin nCp Fg FG ri MEFs in or 6F,G) (Fig. CypD in obtained in were deficient results completely Similar 6D,E). of MEFs (Fig. ability MEFs the wild-type affect in not did CypD of either Depletion levels. protein in sesti,w nce onCp,akyatvtro h MPT a in the resulting of 6C), activator (Fig. key MEFs a wild-type CypD, in down To pore knocked pore. MPT we indicating the this, 6A,B), of assess independent (Fig. is MEFs necrosis PARP1-induced wild-type that in fluorescence calcein Neither MPT. of indicative b is fluorescence calcein mitochondrial EERHARTICLE RESEARCH 4136 the using signaling MPT the of of induction mitochondrial part the was assessed another calcein-Co PARP1-driven pore, first in MPT whether We role the mechanism. examined a necrosis, play of next to mediator appear we not did necrosis, Bax that Given necrosis for induced required not is MPT PARP1- for required Taken not MEFs. 5F,G). is in (Fig. Bax necrosis that overexpression mediated indicate Bcl2 data these by together, protein attenuated deficient Bcl2-encoding were Bcl2 not that a was in cells Bak, the and increase Bax to with both similar for significant However, MEFs 5E) a (Fig. wild-type levels in parallel overexpressing resulted of a by adenovirus activation took Infection Bax therefore inhibited Bcl2. We we the system. where altered have approach the could and which transformed of of one were Bax-dependency either lines Bak, lacking were cell also We particular were 5C,D). these (Fig. that counterparts concerned wild-type their to magnitude LpcoenrMN lctdaysgiiatrdcinin reduction significant any elicited MNNG nor -Lapachone b Lpcoeo NGt oedpnetyidc necrosis induce dose-dependently to MNNG or -Lapachone 2+ loecnemto,weeyardcinin reduction a whereby method, fluorescence b Lpcoe n NGidcdnecrosis MNNG-induced and -Lapachone- b LpcoeadMNNG- and -Lapachone , 5 reduction 85% rae ihteATlgn ogrkcai (supplementary acid bongkrekic ligand S2A,B). ANT Fig. material the with treated yBPAA rCp4kokonddntpeetAIF AIF that prevent confirm not to both wanted for did necessary next indeed knockdown was We Capn4 7E,F). (Fig. or translocation BAPTA-AM by ol lc api ciain N niiin oee,ddnot inhibition did however, JNK AIF inhibition, whether JNK blocked examined activation. inhibition calpain therefore block We rather calpain, could parallel series. in not in act but mediators than these JNK, that suggested that translocation fact The b Ca 7A–D), of inhibition (Fig. against contrast, translocation direct effects In AIF protective cytotoxicity. MNNG induce its with to consistent compounds ability both the 7A,B), blocked of (Fig. completely almost SP600125 AIF Importantly, 2011). inhibitor 7C,D). JNK al., of al., the (Fig. manner et translocation et concentration-dependent a Park nuclear in both Moubarak again 2011; the al., 2006; induced that et MNNG al., Chiu revealed 2010; et al., Immunocytochemistry PARP1- et (Xu in event Artus pivotal death 2007; the be necrotic to thought induced is inter-membrane nucleus mitochondrial the the to space from AIF of Ca translocation not The but JNK through translocation b rMN oidc ellrncoi Fg 8B,C). (Fig. necrosis either cellular of induce ability to siRNA the MNNG on effect or AIF-specific no had an this surprise our with by levels MEFs AIF reduced of Transfection necrosis. ciaintruhidpnetpathways independent through activation LpcoeadMN nueJKadCa and JNK induce MNNG and -Lapachone AIF mitochondrial–nuclear induce MNNG and -Lapachone i.2. Fig. ocnrtoso NGfr4h D etr ltigfrRP nwild- in RIP1 for blotting Western (D) increasing h. ( with 4 type treated for and MNNG MEFs siRNA of in RIP1 staining, concentrations Iodide and Propidium control by with measured as transfected of Necrosis, and (C) concentrations h. control increasing 4 with with for transfected treated MEFs and in measured siRNA as staining, RIP1 for Necrosis, (PI) (B) blotted Iodide control. western Propidium loading then a by and as used h was 48 GAPDH for RIP1. siRNA (RIP1si) RIP1-specific or RIP1. erss smaue yP tiig in staining, displaying PI cells by of Percentage measured (E) as control. necrosis, loading a as used was GAPDH hw r ersnaieo oridpneteprmnspromdin mean performed results are experiments The Results independent h. four duplicate. 4 of for representative MNNG are of shown concentrations increasing with treated F erss smaue yP tiig in staining, PI by measured as Necrosis, of (F) concentrations increasing with treated ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal Ripk1 A Eswr rnfce ih10n fete oto (CONsi) control a either of nM 100 with transfected were MEFs (A) b Lpcoe n NGidcdncoi sidpnetof independent is necrosis MNNG-induced and -Lapachone- +/+ n I1dfcet( RIP1-deficient and ) , 0 Fg A.Hwvr eymc to much very However, 8A). (Fig. 90% 6 s.e.m. b Lpcoe n MNNG-induced and -Lapachone- Ripk1 2 Ripk1 b / lpcoefr4h. 4 for -lapachone 2 Ripk1 2+ T-rnfre MEFs. 3T3-transformed ) /calpain +/+ +/+ and b 2+ b and Lpcoeand -Lapachone Lpcoeand -Lapachone /calpain Ripk1 Ripk1 b b -Lapachone -Lapachone 2 2+ / 2 2 /calpain / 2 MEFs MEFs

Journal of Cell Science fJK a h rdce euto ncl et nue by induced death additive cell in had reduction predicted calpain the and had JNK2 Knockdown we JNK2 necrosis. of PARP1-mediated Finally, of against effects inhibition protective 9C). (Fig. whether phosphorylation tested JNK block to failed MNNG-induced knockdown Capn4 by pathway EERHARTICLE RESEARCH NG(i.9,) ovrey niiino h Ca the of either inhibition by Conversely, 9A,B). induced (Fig. activity MNNG calpain attenuate ncdw fCp4(i.9,) gi niaigta these that indicating parallel. again additional in 9D,E), the operating (Fig. are with pathways Capn4 enhanced However, of significantly above. knockdown shown was as protection 9D,E), this (Fig. MNNG and Lapachone b Lpcoe and -Lapachone- b Lpcoeor -Lapachone 2+ /calpain b - nue yteete ftePR1atvtr.Ipraty we necrosis Importantly, in activators. AIF PARP1 and the MPT of Bax, either RIP1, the for by role protease induced studies, no acting previous the find is unlike it could However, of that pathway. we but JNK activation death the to cell that parallel for in crucial (2) also is and PARP1- activation for calpain necrosis, required demonstrate is cell JNK findings kinase stress induced Our be the not of another. activation might (1) one or JNK, that might to they (RIP1, how coupled and necrosis functionally AIF) and various PARP1-induced MPT the Bax, of of calpain, roles the mediators tested thoroughly proposed we study, present the In DISCUSSION smaue yStxGensann,i oto,JK n JNK2 of and concentrations JNK1 increasing control, with in treated staining, MEFs Green necrosis, siRNA-transfected was displaying Sytox GAPDH cells by of JNK1/2. measured Percentage for (E) as blotted control. loading western a then as and used (JNK2si) h JNK2-specific 48 or for (JNK1si) siRNAs JNK1-specific (CONsi), or control with a h, 4 for in MNNG 20 staining, of without Green concentrations increasing Sytox with by treated measured MEFs as Necrosis, increasing (C) with SP600125. treated MEFs in of staining, concentrations Green (20 Sytox SP600125 by inhibitor measured JNK the without concentrations or increasing with treated MEFs of in JNK total and (pJNK) activation. JNK on 3. Fig. NGfr4h h eut hw r ersnaieo he rfour or are three Results of duplicate. representative mean are in shown performed results experiments The independent h. of 4 JNK2 concentrations for and increasing MNNG JNK1 with as control, treated necrosis, in MEFs displaying staining, siRNA-transfected cells Green of Sytox Percentage by (F) measured h. 4 for Lapachone ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal b Lpcoe(pe aes rMN lwrpnl)fr2hwith h 2 for panels) (lower MNNG or panels) (upper -Lapachone 6 ...* s.e.m. b LpcoeadMN-nue erssi dependent is necrosis MNNG-induced and -Lapachone m P015 D Eswr rnfce ih10n of nM 100 with transfected were MEFs (D) SP600125. M P , b .5vru eil rCONsi. or vehicle versus 0.05 lpcoefr4h iho ihu 20 without or with h, 4 for -lapachone A etr ltigfrpopoyae JNK phosphorylated for blotting Western (A) m ) B erss as Necrosis, (B) M). m M 4137 b -

Journal of Cell Science EERHARTICLE RESEARCH 4138 or MNNG used we whether results same the obtained ake l,21) lhuhwehrRP sas novdin involved also is RIP1 whether although 2011), (Chiu al., process et this being also Park as of reported has been part for study has activation being crucial recent JNK Similarly, as more 2011). al., JNK A et and PARP1. RIP1 hence induced implicated necrosis and for responsible MNNG, being by as is events of of sequence what AIF ablation is Indeed, PARP1 compound. either effects. by activate induced cytotoxic to their Parp1 ability for indicate common would responsible This their activators. PARP1 that distinct two Lapachone, ue l X ta. 06 aepooe RIP1–JNK–MPT– a proposed have 2006) al., et (Xu al. et Xu h AP-noiggn,efcieyaoihddeath abolished effectively gene, PARP1-encoding the , b Lpcoeidcdcl et Sihe l,1999; al., et (Shiah death cell -Lapachone-induced b - ta. 02.Hwvr ehv on httepoetv effects protective the that found been have we has (Jouan-Lanhouet However, 2012). necrosis as al., TRAIL-induced et pathway for in reported RIP1 necroptotic of recently with downstream necrosis. RIP1–RIP3-dependent actually is inhibitor obtained MNNG-induced PARP1 the RIP1 that were is prevent the possibility results One or to negative siRNA failed Similarly with also either acute necrostatin However, RIP1, protein. of another by inhibition overcome of RIP1 was RIP1 upregulation that of loss compensatory such chronic the pathways, or dispensable, the became altered procedure transformation td.Dsieti,w eeual orcptlt hs aain data these recapitulate to unable were 3T3-transformed we this, Despite study. b Lpcoesefcshsnvrbe etdutltepresent the until tested been never has effects -Lapachone’s ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal eedn nCa on dependent 4. Fig. yo re tiig nMF rae ihincreasing with treated of MEFs concentrations in staining, Green Sytox xeiet efre ndpiae eut r mean are Results * independent duplicate. four in results or performed The three experiments h. of 4 representative for are MNNG shown treated of Green MEFs concentrations Sytox siRNA-transfected increasing by Capn4 with measured and as control Necrosis, in (G) staining, h. of 4 concentrations for increasing lapachone with siRNA- treated Capn4 and MEFs as control Necrosis, transfected in (F) staining, control. Green for Sytox loading blotted by a western measured as then used Capn4- and was or h GAPDH (CONsi) 48 Capn4. control for a siRNA were either (CAPN4si) MEFs of specific (E) nM with h. 100 treated 2 with MEFs for transfected in MNNG activity of Calpain concentrations (D) increasing of h. concentrations 2 increasing for with Lapachone 1 treated without MEFs or in with activity of h, concentrations 4 increasing for with MNNG Green treated Sytox MEFs by in measured staining, as Necrosis, (B) BAPTA-AM. P , .5vru eil rCONsi. or vehicle versus 0.05 b Ripk1 LpcoeadMN-nue erssis necrosis MNNG-induced and -Lapachone 2 / 2 b 2+ Lpcoefr4h iho ihu 1 without or with h, 4 for -Lapachone Es fcus ti osbeta the that possible is it course Of MEFs. n calpain. and m A erss smaue by measured as Necrosis, (A) AT-M C Calpain (C) BAPTA-AM. M b -Lapachone. 6 s.e.m. m b M - b -

Journal of Cell Science EERHARTICLE RESEARCH on hti a h N2ioomta predominantly that isoform JNK2 the we and siRNA was MNNG- Using both 2011). it to al., contributed than et that rather (Park JNK2 important found was was it MNNG. that that to indicated JNK1 resistant study another were al., et contrast, cells JNK (Xu In JNK1-deficient al. and et that translocation Xu pharmacological found activators. AIF PARP1 2006) the both and by blocking elicited necrosis in translocation, cell efficient AIF was inhibition JNK induced activated dose-dependently and compounds Both cytotoxicity. and MNNG- 2014). both al., could et RIP3 (Sosna or necrosis block RIP1 MNNG-induced to of prevent fails inhibition not PARP1 our that of and inhibition with necrosis that Consistent TNF-induced found al. pathways. in et do Sosna necrotic they results, separate that suggesting represent shown), oxidative-stress-induced not fact against (data additive inhibition are PARP1 necrosis and RIP1 of ihol io otiuinfo N1 h exact The JNK1. activation from JNK to contribution leads activity minor PARP1 which a by mechanisms only with ncnrs oRP,w i ofr htJKi eurdfor required is JNK that confirm did we RIP1, to contrast In b LpcoeidcdAFtasoainand translocation AIF -Lapachone-induced b Lpcoeidcdnecrosis, -Lapachone-induced eaieMK1cudas lc eldaheiie by dominant- elicited and death 2000), cell al., block et also (Parra could MKK7 MEKK1 negative and dependent is MEKK1 activation JNK on MNNG-induced clear. entirely not is 04.Hwvr efudn vdneta P sanecessary a is al., MPT et that evidence in (Dodoni no MPT found PARP1 induce we of However, directly 2004). independent could al. MNNG mitochondria, et that Dodoni isolated 2005). indicated al., et also Schinzel the al., have 2005; et of al., (Baines et stimuli component PARP1- necrotic Nakagawa regulatory Other to 2005; in resistant major cells 2006). and ultimately MPT renders a 2004), pore, al., CypD, for MPT al., process et of role et (Alano ablation the a necrosis (Xu indeed and such was translocation AIF suggested AIF MPT of induced also release that have the studies indicated are for al. mechanisms responsible et indirect of Xu more ADP-ribosylation direct other unknown. involves whether is the involved this or that Whether indicate MEKK1 upstream further would JNK. much point This some of at 1999). acting are al., activators PARP1 et (Shiah Lapachone stels tpi h rpsdPR1RP–N1pathway, PARP1–RIP1–JNK1 proposed the in step last the As ramn ih30n tuoprn o 8hwsue sapositive a as ( used 50 was bar: h Scale 18 control. for staurosporine nM 300 with Treatment and 20 either with treated Bax. of independent 5. Fig. C erss smaue yStxGensann,in staining, Green control. Sytox loading by measured a as as Necrosis, used (C) was GAPDH MEFs. transformed tiig in staining, b xeiet efre ndpiae eut r mean independent are four Results * of duplicate. representative in are performed for shown experiments MNNG results of The concentrations h. increasing 4 with in treated staining, MEFs Green infected Sytox by measured as Necrosis, of (G) concentrations increasing in Necrosis, with (F) staining, treated GAPDH. Green MEFs and Sytox Bcl2 by for blotted measured western as then and infected h were 48 for MEFs either (E) for h. encoding 4 adenoviruses with for MNNG of concentrations increasing Bax P Lpcoefr4h D erss smaue yStxGreen Sytox by measured as Necrosis, (D) h. 4 for -Lapachone ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal , .5versus 0.05 Bax +/+ /Bak b 2 LpcoeadMN-nue erssis necrosis MNNG-induced and -Lapachone / 2 +/+ Bax / Bak and ) +/+ 2 Bax / / 2 Bak Bax Estetdwt nraigcnetain of concentrations increasing with treated MEFs +/+ m m A muotiigfratv a nMEFs in Bax active for Immunostaining (A) +/+ /Bak M .()Wsenbotn o a nwild-type in Bax for blotting Western (B) m. 2 / and 2 b / lpcoeo MMN o h. 2 for MNNG mM 1 or -lapachone +/+ Bak . Bax 2 / 2 2 / BxadBkdfcet SV40- deficient) Bak and (Bax 2 / b Bak glcoiae( -galactosidase 2 b / a-o Bcl2-infected or Gal- 2 Estetdwith treated MEFs b Lpcoefr4h. 4 for -Lapachone b Bax 6 b a-o Bcl2- or Gal- a)o Bcl2 or Gal) s.e.m. +/+ / Bak 4139 +/+ b -

Journal of Cell Science boaetencoi lctdb NGand MNNG by elicited necrosis the abrogate EERHARTICLE RESEARCH hsi ossetwt idnsta hlto fCa of 4140 chelation 2011). that al., findings cytotoxicity et Chiu with PARP1-induced 2009; consistent al., mediating et is Vosler crucial in 2003; This a al., reported calpain et necrosis have (Tagliarino papers and for several translocation 2007), role al., AIF et MNNG-induced (Moubarak being as for group Susin’s essential by proposed First al., 2011). controversial. al., somewhat et et Abeti (Liu 2010; necrosis al., et both PARP1-mediated the Loor are 2002; blocking is that deletion CypD in demonstrating This and ineffective cyclosporine-A studies necrosis. inhibitor previous MPT, pore PARP1-mediated MPT other inhibits block also with to which consistent acid, with failed treatment bongkrekic similarly However, inhibitor opening. ANT pore CypD- the the MPT bypassing of simply step were concern activators dependent of One PARP1 agents. the deletion these that to (knockdown) response was in acute death cell nor affected CypD or (knockout) MNNG- chronic neither either in step h oeo api nmdaigPR1idcdncoi is necrosis PARP1-induced mediating in calpain of role The b Lpcoeidcdncoi and necrosis -Lapachone-induced b -Lapachone 2+ can hlto a oefc nmtcodilt-ula AIF This activators. mitochondrial-to-nuclear PARP1 Ca the the on of activation of JNK, unlike either effect that, suggests by no evoked translocation had chelation ob eesr o AP-nue erss oee,Ca However, necrosis. PARP1-induced for necessary be to ihteJKihbtr ovreyihbto fclanddnot did activation calpain calpain of block inhibition to results conversely our inhibitor; unable why JNK also sure the were not with are We we different. and are in reports are previous results These to translocation. contrast and release AIF for dispensable AT ol infcnl euebt NG and MNNG- both reduce Importantly, significantly MEFs. and in MNNG could activity both BAPTA calpain that increased found dependently we regulatory small study, that, the and lacking toxic m- indicated MEFs of its in subunit has activity, abrogated 2011). calpain not group al., elevate were et effects Dawson’s indeed Chiu could from 2006; MNNG al., report although et a Bentle 2001; However, al., et (Tagliarino ncigdw h ml api uui.Tu,a es nour in least at Ca Thus, the subunit. of calpain activation by small hands, phenocopied the was down that knocking finding a necrosis, Lapachone-induced ihicesn ocnrtoso NGfr4h F etr blotting ( Western wildtype (F) h. in 4 CypD treated for for MEFs MNNG of siRNA-transfected concentrations CypD increasing and with control in staining, PI increasing with in treated of staining, MEFs concentrations (PI) siRNA-transfected control. Iodide RIP1 loading Propidium and a by control as measured used as was Necrosis, then GAPDH and (D) h CypD. 48 for for blotted siRNA h. western (CypDsi) 2 CypD-specific for or (CONsi) MNNG control mM a 1 or 50 vehicle bars: to Scale exposed MEFs in fluorescence by determined (MPT) calcein/CoCl transition permeability Mitochondrial transition. (A) permeability mitochondrial of independent 6. Fig. aahn o .()MTdtrie ycalcein/CoCl by determined MPT (B) h. 2 for lapachone ulct.Rslsaemean in are are performed Results shown experiments duplicate. results independent The four h. of 4 representative for MNNG of concentrations of concentrations AD a sda odn oto.()Ncoi,a measured as Necrosis, (G) in control. staining, loading PI a by as used was GAPDH yP tiig in staining, PI by ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal b LpcoeadMN-nue erssis necrosis MNNG-induced and -Lapachone 2 loecnei Esepsdt eil r20 or vehicle to exposed MEFs in fluorescence m m .()MF eetasetdwt 0 Mo either of nM 100 with transfected were MEFs (C) m. clan Wn ta. 09.I h present the In 2009). al., et (Wang -calpains Ppif Ppif b b lpcoefr4h E erss smaue by measured as Necrosis, (E) h. 4 for -lapachone Lpcoefr4h H erss smeasured as Necrosis, (H) h. 4 for -Lapachone +/+ +/+ Ppif 2+ and and +/+ clansse oside appear indeed does system /calpain n yDdfcet( CypD-deficient and ) 6 Ppif Ppif ...* s.e.m. 2 2 / / 2 2 Estetdwt increasing with treated MEFs increasing with treated MEFs P , .5versus 0.05 b 2+ Lpcoedose- -Lapachone clansse is system /calpain Ppif Ppif 2 / 2 2 MEFs. ) +/+ . m M b b 2+ - -

Journal of Cell Science EERHARTICLE RESEARCH fee ykokono N2adCp4wsadditive. was Capn4 and protection JNK2 that of finding knockdown the by was of in notion offered than this mediators rather with important parallel Consistent in are series. acting calpain are they and indicate necrosis, JNK PARP1-induced would data both these although together, that Taken activation. JNK affect a nteMF.Hwvr niiino a,ete through either ability the Bax, affect not of did Bcl2, inhibition of overexpression However, or targeting MEFs. gene the found We in Bax. Bax for and role MNNG a both confirm that not could we necrosis, induced ept orbrtn oefrJKadclani PARP1- in calpain and JNK for role a corroborating Despite eedn nJKbtntCa not but JNK on dependent 7. Fig. MMN o ,wt rwtot20 without or with h, 2 for MNNG mM 1 20 20 with treated MEFs in AIF NGfr2h iho ihu 1 without or of with h, concentrations 2 increasing for with MNNG treated MEFs in translocation 1 without or of with concentrations increasing or with with treated h, MEFs 2 for MNNG 20 of without concentrations increasing with treated 20 of concentrations increasing 50 o .Terslssonaerpeettv ftreo four or are three Results of duplicate. mean representative in are performed shown experiments results independent MNNG The of h. siRNA- concentrations 2 Capn4 increasing for and with control treated in MEFs translocation transfected AIF of Quantification of (H) MEFs concentrations siRNA-transfected increasing Capn4 with and treated control in translocation AIF ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal m m m P015 B muotiigfrAFi Estetdwith treated MEFs in AIF for Immunostaining (B) SP600125. M P015 D uniiaino I rnlcto nMEFs in translocation AIF of Quantification (D) with SP600125. treated M MEFs in translocation AIF of Quantification (C) m. 6 ...* s.e.m. b Lpcoe n NGidcdAFtasoainis translocation AIF MNNG-induced and -Lapachone- m P015 E uniiaino I rnlcto in translocation AIF of Quantification (E) SP600125. M P , m .5vru vehicle. versus 0.05 AT-M F uniiaino AIF of Quantification (F) BAPTA-AM. M b Lpcoeidcdaml ciainof activation mild a induced -Lapachone m b M lpcoefr2h iho without or with h, 2 for -lapachone m b 2+ AT-M G uniiainof Quantification (G) BAPTA-AM. M Lpcoefr2h iho without or with h, 2 for -Lapachone /calpain. m b P015 cl bars: Scale SP600125. M lpcoefr2h. 2 for -lapachone A muotiigfor Immunostaining (A) b lpcoefr2h, 2 for -lapachone 4141

Journal of Cell Science EERHARTICLE RESEARCH uhta,freape I1adAFbcm novdi the in 4142 involved in become dispensable are AIF they cells whereas and activation, transformed PARP1 RIP1 the of effects example, in toxic altered for that, are pathways such be signaling might technical our It the ones. is immortalized acute more that study of versus our cells more cultured in a effects primary noted of the others Alternatively, cytotoxic use the for and study. discrepancy chronic necessary this our for the not reason might in it is for observed that but required effects shown). is activators possibility is (not PARP1 One clear. AIF the time not that each is be this results different for similar two reason tried obtained The actually or and MNNG- We either siRNAs block death. to cell cell sufficient that not Lapachone-induced finding PARP1-driven was the was AIF for surprise of our depletion prerequisite to much a very However, necrosis. is translocation MEFs nuclear in death 2001). MNNG-induced Duerksen-Hughes, block and of (Yang can inhibitor efflux an AIF ceramide synthesis and 2009) induce al., ceramide Alternatively, can et (Scharstuhl ceramide 2012). mitochondria and as the (Lei from al., involved, JNK be et by might upregulated channels Chaanine proteins and/or 2003; these activated of be Davis, both to Moreover, known can 2010). MPT-independent are BNIP3 al., and an et 2011), (Quinsay in al., manner permeabilization et Kim mitochondrial and 2008; induce al., Bmf TNF-induced et of example, (Hitomi mediators as For necrosis is proposed been responsible. JNK recently be have how BNIP3 might of to members question response family in those the or mitochondria with the raise MNNG from coupled MPT, either AIF of data, for release the role these inducing a way death the out Either for were required ruling calpain. not was of events this (Byun but effects cells, activation induced our two have in PARP1 might activation activation be of Bax calpain the might Alternatively, independently 2009). activators al., Bax that et PARP1 induce as and to indicating interesting able MNNG is by shown), This Bax death, independent. not of cell blocked activation (data which block SP600125, not found gene- with also did we inhibition The finding this JNK death. with that compensation Consistent necrotic any protein. out related evoke ruling this thus by Bak, to lacked also activators MEFs targeted PARP1 the of l h bv speiae ntecnetta I ees and release AIF that concept the on predicated is above the All b Lpcoe ti luil htohrBcl2 other that plausible is It -Lapachone. b -Lapachone b - o-rnfre el.Ti ol nfc evr neetn sit different as is interesting signaling PARP1-driven very that be indicate potentially fact would in would This cells. non-transformed fteeezmsi oelkl ob fciia eei than benefit clinical of be to likely individually. one more each is targeting both enzymes of the targeting mediate these dual to Thus, parallel of activation. in PARP1 act of calpain effects and necrotic JNK both Instead that death. appears cell it necrotic PARP1-induced acute for dispensable mediate to necrosis. our available in protein proteins sufficient various left efficient, the although of study, knockdowns the that conceivable Finally therapeutically. is exploited be it could that fact a cells, cancer in aaa.MF eeifce ihteaeoiu tamlilct of multiplicity a at adenovirus Manitoba, the with Winnipeg, infected Research, were MEFs Hospital Canada). Lorrie from (St-Boniface gift generous a Kirshenbaum was Bcl2 for adenovirus FBS, Replication-deficient siRNAs (v/v) and 10% Adenovirus mg/ml). with (0.1 streptomycin supplemented and were Cells U/ml) medium Korsmeyer. (100 Stanley DMEM penicillin late the in from gift maintained generous a were MEFs care wild-type, and cultured use the Primary for guidelines animals. NIH the and of to Care conformed embryos Animal and Missouri-Columbia and Committee of tissues Use University mouse the of by approved harvesting were the involving experiments All culture Cell from purchased penicillin/ were (HBSS), (FBS) serum solution bovine saline fetal Hyclone; buffered and were streptomycin Hanks’s RNAiMAX medium Lipofectamine Eagle’s modified (DMEM), and Dulbecco’s Technologies; Life Green from purchased Sytox Iodide, Propidium Reagents METHODS AND MATERIALS eeotie rmE351. mro ytysndgsinas 2011). digestion Baines, and trypsin McGee by 2005; and al., embryos wild-type 3T3-transformed et The E13.5–15.5 (Baines described from previously obtained were rmMcel elhr(nvriyo ascuet eia School, and Medical wild-type SV40-transformed Massachusetts The of MA). (University Worcester, Kelliher Michelle from NGwsfo hmSrie l te hmcl n egnswere reagents and chemicals other All Sigma-Aldrich. Service. Promega; from from Chem purchased from were was assay MNNG activity calpain-GLO the zVAD- and Axxora; from FMK purchased were ester) (acetoxymethyl BAPTA-AM nsmay efudta I1 I,BxadMTare MPT and Bax AIF, RIP1, that found we summary, In ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal NGfr4h h eut hw r ersnaieo six of Results mean representative duplicate. are are in shown performed results experiments The independent h. of 4 siRNA- concentrations for AIF increasing MNNG and with control treated in MEFs staining, transfected Green Sytox by measured increasing with in treated of staining, MEFs concentrations Green siRNA-transfected Sytox loading AIF by a and measured as control as used Necrosis, was and (B) GAPDH h AIF. control. 48 for for blotted siRNA western (AIFsi) AIF-specific then or (CONsi) control a either for necrosis. dispensable induced is AIF 8. Fig. b Lpcoe P015 ersai-,bnkei cdand acid bongkrekic necrostatin-1, SP600125, -Lapachone, 6 s.e.m. b Lpcoefr4h C erss as Necrosis, (C) h. 4 for -Lapachone A Eswr rnfce ih10n of nM 100 with transfected were MEFs (A) Ripk1 b Lpcoe n MNNG- and -Lapachone- 2 Ppif / 2 2 Eswr eeosgift generous a were MEFs / 2 and , Parp1 Bax 2 2 / 2 / 2 /Bak MEFs 2 / 2

Journal of Cell Science oto iN,wr bandfo ieTcnlge.Posof Pools Technologies. Life (5 from JNK1 obtained against duplexes were siRNA siRNA, control GGGAUGCUGCAUGCUUCUAUGAUAU-3 EERHARTICLE RESEARCH neto MI f3 lqefriguisfr4 .TesiRNA The h. 48 (5 for Capn4 units (5 RIP1 plaque-forming AUAA-3 mouse 30 against of duplexes (MOI) infection 9 9 ,Cp (5 CypD ), -ACUGACCGAUCAGGGACUAUCGGUA-3 9 -UCACAGACUGUGGCCAGUUGAGUA-3 9 -GGGCCAGUAGCAGAUGACCUC- 9 -GAAACUACAACCAACAGUA-3 9 ,a ela non-specific a as well as ), 9 ,adAF(5 AIF and ), 9 9 ), 9 - , 0 MsRAuigLpfcaieRAMXadte utrdfor cultured experiments. then various the and to RNAiMAX prior Lipofectamine h 48 using siRNA nM 100 CAA-3 5 UUAAGUCGUCCUUUU-3 AGA-3 5 9 9 -GCAUUCAGCUGGUAUCAUU-3 -GAUUGGAGAUUCUACAUUC-3 9 9 eeotie rmDamcn Eswr rnfce with transfected were MEFs Dharmacon. from obtained were ) ,5 ora fCl cec 21)17 1444 doi:10.1242/jcs.128009 4134–4145 127, (2014) Science Cell of Journal ocnrtoso NGfr2h iho ihu 20 without or with increasing h, with 2 treated for MNNG MEFs of in concentrations activity Calpain (B) SP600125. one of of concentrations independent are activation calpain another. and JNK 9. Fig. eitn AP-nue necrosis. PARP1-induced mediating lutaigtepooe aallJK n Ca and JNK2 parallel proposed mean the are illustrating Results experiments duplicate. independent in are four performed shown or results three The h. of with 4 representative treated for MNNG then of and concentrations with siRNAs increasing transfected JNK2+Capn4 MEFs or in JNK2, staining, control, Green Sytox by with measured treated then of and concentrations with siRNAs increasing transfected JNK2+Capn4 as MEFs or Necrosis, in JNK2 (D) staining, control, h. Green 2 Sytox for by panels) measured (lower MNNG or of panels) concentrations increasing MEFs with siRNA-transfected treated (CAPN4si) JNK Capn4 total and and (CONsi) (pJNK) control JNK in phosphorylated for blotting Western (C) 9 -GGAAAGAACUGAUAUACAA-3 A api ciiyi Estetdwt increasing with treated MEFs in activity Calpain (A) b Lpcoefr2h iho ihu 20 without or with h, 2 for -Lapachone 9 ,5 9 -ACUAAUGGAUGCUAACUUA-3 b Lpcoefr4h E erss as Necrosis, (E) h. 4 for -Lapachone 9 9 ,5 ,5 9 9 -GGAAAGAGCUAAUUUA- -GGAGUUAGAUCAUGAA- 9 n N2(5 JNK2 and ) b 6 Lpcoe(upper -Lapachone ...()Schematic (F) s.e.m. 2+ clanpathways /calpain m SP600125. M 9 -GAAG- m M 4143 9 ,

Journal of Cell Science a osdrdsaitclysignificant. statistically considered was Bax yo re nHS o 0mna 37 at min 30 for HBSS in Green Sytox or Iodide Propidium either with incubated were plates 12-well on cells Briefly,treated 2011). Baines, and McGee 2005; as al., exclusion, et Green (Baines described Sytox previously or Iodide Propidium by determined was Necrosis assays Fluorescence ARTICLE RESEARCH 4144 R. M. Duchen, and Y. A. Abramov, R., Abeti, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.128009/-/DC1 online available material Supplementary material Supplementary months. 12 after release for PMC in Deposited C.P.B.]. HL092327, numbers to [grant HL094404 Health HL094404, of Institutes National by supported was work This Funding manuscript. the wrote C.P.B. data. the the conducted analyzed C.P.B. and and D.L.D. experiments experiments. the designed and conceived C.P.B. contributions Author interests. competing no declare authors The interests Competing the of gifts generous Stanley their Kelliher, for Kirshenbaum Michelle Lorrie for thank and MO) also Korsmeyer Columbia, We Missouri-Columbia, assistance. of technical (University excellent Crombie Kathryn thank We Acknowledgements Student’s the by calculated was significance Statistical by visualized analyses Statistical and Biotechnology) Cruz alkaline-phosphatase-linked (Amersham). chemifluorescence appropriate (Santa enhanced Biotechnology; the antibody Cruz with BD Membranes secondary Santa incubated Abcam. from from from then CypD Bcl2 and were AIF Millipore; and from caspase-3 GAPDH and Bax and cleaved PVDF Capn4 Signaling; RIP and Cell JNK available proteins: onto Promega; from commercially from following following phospho-JNK transferred the Biosciences; the against acrylamide, using by resolved antibodies blotted were 10–15% Proteins Tris-HCl and X-100. mM membranes, Triton 10 using 1% NaCl, and mM EDTA, SDS-PAGE 150 mM containing 1 buffer 7.4, pH in lysed were Cells blotting inverted Western an with camera. digital collected a were to connected cells (Alexa IX51) (Olympus the antibody microscope fluorescence of secondary Images TRITC-conjugated 1:500). or Invitrogen, with FITC- incubated then respective were Cells 1:100). the 6A7, clone form Biosciences against active (BD the antibody recognizes Bax that of antibody an an or with 1:500) Biosciences, either (BD AIF stained and blocked, paraformaldehyde, b Immunocytochemistry CaCl mM 2 to response in and baseline at both activity, Calpain assay activity Calpain h eutn loecneiae eecletduiga inverted an camera. using digital 1 with incubated were a MEFs collected MPT, of determination to the For connected were IX51) images (Olympus microscope fluorescence fluorescence resultant The nuclei. all stain to bis-benzamide with counterstained and paraformaldehyde rmg.Atvt a acltda h ifrnebtentebsland from basal the assay between difference activity the Ca as luminescence calculated was Calpain-GLO Activity Promega. the using determined loecnemcocp OypsI5)cnetdt iia camera. digital a to connected IX51) (Olympus microscope fluorescence inverted an using collected fluorescence images and HBSS with twice washed ls1m CoCl mM 1 plus Lpcoe rMN-rae Eswr ie ih4% with fixed were MEFs MNNG-treated or -Lapachone- asn srctcmtblcfiueadnuoa death. neuronal and failure metabolic astrocytic causing 2+ 2 idcdactivities. -induced / 2 / Bak 2 / 2 Es n c2aeoiu,respectively. adenovirus, Bcl2 and MEFs, 2 nHS o 0mna 37 at min 30 for HBSS in ˚ .Clswr hnfxdi 4% in fixed then were Cells C. (2011). ˚ .Delae el eethen were cells Dye-loaded C. b ayodatvtsPARP activates -amyloid Brain m Ripk1 134 t calcein-AM M -test. 1658-1672. , 2 / 2 P MEFs, , 2 was , 0.05 rik,K . allnaaa,K,Taa .J,Cadaorh,H C., H. Chandramoorthy, J., R. Thapa, K., Mallilankaraman, M., K. and Irrinki, J. R. Xavier, A., Degterev, J., Yao, A., Ng, E., D. Christofferson, J., Hitomi, e . ag . io . ag . u . ho .adWn,X. Wang, and L. Zhao, F., Du, T., Wang, L., Miao, L., Wang, S., He, og . e,E . i .S,Kbai . a,J,Xe .J,Hih .T,Gao, T., J. Hsieh, J., X. Xie, J., Yan, W., Kabbani, S., L. Li, A., F. E. Bey, Lisa, Y., Dong, Di and P. Bernardi, V., Petronilli, M., Canton, G., Dodoni, Chan, and M. Guildford, D., T. Ray, R., Genga, D., Moquin, S., Challa, S., Y. Cho, as,P O. P. Hassa, hu .Y,H,F . ha,S . hn,Y n i,W W. W. Lin, and Y. Chang, G., S. Shiah, M., F. Ho, Y., L. Chiu, E. R. Gordon, K., Fish, R., E. Chemaly, L., Liang, D., Jeong, H., A. Chaanine, obrk .S,Yse .J,Ats . ohror . re,P A., P. 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