Inactivation of the ferroptosis regulator Gpx4 …...preventing lipid-oxidation-induced acute renal...

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Inactivation of the ferroptosis regulator Gpx4 triggersacute renal failure in miceJose Pedro Friedmann Angeli1, Manuela Schneider2, Bettina Proneth1, Yulia Y. Tyurina3, Vladimir A. Tyurin3,Victoria J. Hammond4, Nadja Herbach5, Michaela Aichler6, Axel Walch6, Elke Eggenhofer7,Devaraj Basavarajappa8, Olof Rådmark8, Sho Kobayashi1,9, Tobias Seibt1, Heike Beck10, Frauke Neff6,Irene Esposito11, Rüdiger Wanke5, Heidi Förster1, Olena Yefremova1, Marc Heinrichmeyer1,Georg W. Bornkamm12, Edward K. Geissler7, Stephen B. Thomas13, Brent R. Stockwell13, Valerie B. O’Donnell4,Valerian E. Kagan3, Joel A. Schick1 and Marcus Conrad1,14

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cellsoverexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here,we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologicallyrelevant form of ferroptosis. Using inducible Gpx4−/− mice, we elucidate an essential role for the glutathione/Gpx4 axis inpreventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library ofsmall molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative calledLiproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4−/− mice, and in a pre-clinical model ofischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of celldeath, which, when impeded, promises substantial cytoprotection.

Previously it was assumed that apoptosis was the only regulated formof cell death. Yet, recent years have shown that non-apoptotic celldeath pathways are highly heterogeneous processes characterized bymorphologically and biochemically distinct events1. Non-apoptoticcell death pathways have gained special attention particularly dueto their role in inflammation and as potential therapeutic target inapoptosis-resistant tumours2,3. In this context, at least two cell deathpathways are executed in response to detrimental concentrations ofpartially reduced forms of oxygen (that is, reactive oxygen species4).They include regulated necrosis (that is, necroptosis)5, and an iron-dependent form of cell death, named ferroptosis6. Ferroptosis is anewly discovered cell death pathway that can be induced in a subset

of tumour cell types, including cell lines engineered to overexpressoncogenic RAS, by a number of small molecules named ferroptosis-inducing agents7 (FINs). It is characterized by increased levels of lipidhydroperoxides and iron overload, leading to caspase- andnecrosome-independent cell death. FINs have been classified into class I FINsthat involve cellular glutathione (GSH) depletion and class II FINsthat lack this characteristic. Recently, it has been shown that class IIFINs trigger ferroptosis through inhibition of glutathione peroxidase 4(Gpx4; ref. 8).

So far, exploitation of ferroptosis has been proposed for killingtumour cells in response to specific compounds, and as a potentialmechanism involved in oxidative glutamate toxicity in cells and brain

1Helmholtz Zentrum München, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. 2Institute for Stroke and DementiaResearch, Klinikum der Universität München, Ludwig-Maximilians-University Munich, Marchioninistr. 15, 81377 Munich, Germany. 3Department of Environmentaland Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA. 4Department of Infection, Immunity and Biochemistry, School of Medicine,Cardiff University, Heath Park, Cardiff CF14 4XN, UK. 5Institute of Veterinary Pathology, Center for Clinical Veterinary Medicine, Ludwig-Maximilians-UniversityMunich, Veterinaerstr. 13, 80539 Munich, Germany. 6Institute of Pathology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.7Department of Surgery, University Medical Center of Regensburg, Franz-Josef-Strauss Allee 11, 93053 Regensburg, Germany. 8Department of Medical Biochemistryand Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-17177 Stockholm, Sweden. 9Department of Food and Applied Life Sciences, Faculty ofAgriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan. 10Walter Brendel Centre of Experimental Medicine, Munich Heart Alliance,Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich, Germany. 11Institute of Pathology, Technische Universität München, Ismaningerstr. 22, 81675Munich, Germany. 12Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Zentrum München, Marchioninistr. 25, 81377 Munich, Germany.13Department of Biological Sciences and Department of Chemistry, Howard Hughes Medical Institute, Columbia University, 550 West 120th Street, Northwest CornerBuilding, MC 4846, New York, New York 10027, USA.14Correspondence should be addressed to M.C. (e-mail: [email protected])

Received 13 August 2014; accepted 14 October 2014; published online 17 November 2014; DOI: 10.1038/ncb3064

1180 NATURE CELL BIOLOGY VOLUME 16 | NUMBER 12 | DECEMBER 2014

© 2014 Macmillan Publishers Limited. All rights reserved.

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slices9,10. We report here direct in vivo evidence that this type of celldeath is not only limited to specific tumours treated with FIN agents,but that Gpx4 prevents premature death ofmice by actively restrainingthe ferroptotic machinery in kidney tubular cells. In light of the yet-uncharacterized role of lipid hydroperoxides in ferroptosis, we nowprovide a detailed picture of a complex lipid oxidation signature inresponse toGpx4 inactivation and ferroptosis induction. Furthermore,we identified an in vivo ferroptosis inhibitor shown to be efficaciousin inducible Gpx4−/− cells and mice, as well as tissues subjected toischaemia/reperfusion. This class of small-molecule inhibitors willnot only serve to define the involvement of ferroptotic cell death inpathological conditions, but will also facilitate characterization of theunderlying molecular paradigms that regulate ferroptosis.

RESULTSInducible disruption of Gpx4 causes acute renal failure andearly death in an Alox15-independent mannerWe previously showed that the inducible disruption of Gpx4 in mouseembryonic fibroblast (MEF) cells (Pfa1 cells) leads to a form of celldeath with necrotic-like features that involves 12/15-lipoxygenase(Alox15) and apoptosis inducing factor (AIF) activation11. Accord-ingly, Alox15−/− cells were found to be resistant to the effects of Gpx4inhibition through depletion of GSH by L-buthionine sulphoximine11

(BSO; Fig. 1a). To extend this initial analysis, inducible Gpx4−/− cellswere treated with a series of inhibitors including MJ33, a competi-tive and reversible inhibitor of calcium-independent phospholipaseA2 (aiPLA2), the lipoxygenase inhibitor zileuton, MK886 (Alox5),PD146176 (Alox15) and the dual inhibitor BWA4C (Alox5 and 15),which all prevented cell death in response to Gpx4 deletion (Fig. 1b).

In light of these and our previous data, we sought to challenge thein vivo relevance of the Gpx4–Alox15 axis in this cell death pathway.We generated and characterized mouse models based on mice withloxP-flanked Gpx4 alleles, along with mice proficient or deficient inAlox15 (ref. 12). After 2–3 breeding stepsCreERT2;Gpx4fl/fl /Alox15+/+

and CreERT2;Gpx4fl/fl /Alox15−/− mice were generated, which did notshow any abnormalities in the absence of tamoxifen (TAM). OnTAM feeding, the overall behaviour of CreERT2;Gpx4fl/fl /Alox15+/+

changed markedly, as indicated by catatonia, hunched postureand loss of body weight, whereupon mice had to be euthanized.Unexpectedly, CreERT2;Gpx4fl/fl /Alox15−/− revealed exactly the samebehaviour and had to be euthanized at the same stage as inducedCreERT2;Gpx4fl/fl /Alox15+/+ mice (Fig. 1c).

To define the role of Alox15 in cell death, cellular systems wereestablished from CreERT2;Gpx4fl/fl /Alox15−/− mice (PZL cells) andfrom CreERT2;Gpx4fl/fl /Alox15+/+ mice as control (PZ cells). WhenGpx4 deficiency was induced by TAM (Supplementary Fig. 1A), PZLcells died like PZ cells (Supplementary Fig. 1b), which could notbe rescued by N -acetylcysteine (NAC) but by α-tocopherol (αToc)as previously reported11 (Supplementary Fig. 1b). As 5-lipoxygenase(Alox5) might compensate for loss of Alox15 (ref. 13), Alox5 wasdepleted by short hairpin RNA (shRNA) in PZL and PZ cells(Supplementary Fig. 1c,d). Whereas the knockdown of Alox5 in PZLcells partially delayed the cell death process, it did not impact on celldeath in PZ cells. From these findings we conclude that no individuallipoxygenase could be identified that is solely responsible for the celldeath processes downstream of Gpx4 inactivation.

Inferring that more than one lipid oxygenase is involved in celldeath, we endeavoured to characterize in more detail why animalsdie on Gpx4 disruption. On dissection, grossly, slightly enlarged andpale kidneys were evident in the Gpx4−/− mice (Fig. 1d). In rare cases(<5%), signs of focal liver necrosis were evident. Analysis of Gpx4expression showed a marked decrease of Gpx4 in the kidney alreadyseven days after TAM treatment (Fig. 1e), which was paralleled byprogressive, unselective proteinuria (Fig. 1f) and altered biochemicalkidney parameters (Supplementary Table 1).

Immunohistochemistry demonstrated a strong Gpx4 staining inthe proximal tubules of wild-type kidney that was lost on TAMadministration (Fig. 1g). Histological and ultrastructural analysis aswell as terminal deoxynucleotidyl transferase (TdT)-mediated dUTPnick end labelling (TUNEL) revealed widespread cell death of prox-imal tubular epithelia with progression to effacement of all tubularepithelia and of the parietal epithelial cells of the Bowman’s capsule(Fig. 1h,i and Supplementary Fig. 1f). Proteinaceous casts and cellulardebris were observed in affected tubuli (Fig. 1h), and focal signs oftubular regeneration were evident (Fig. 1i). Mononuclear interstitialinfiltration and edema were observed in advanced lesions (Fig. 1h andSupplementary Fig. 1g). Notably, CreERT2;Gpx4fl/fl /Alox15−/− micedeveloped comparable lesions to those ofCreERT2;Gpx4fl/fl /Alox15+/+

mice (Supplementary Fig. 1e). Immunohistochemical staining againstcleaved caspase-3 revealed faint staining, mainly confined to the lu-men of renal tubules, possibly resulting from secondary inflammatoryprocesses (Supplementary Fig. 1g).

Inducible Gpx4 disruption induces Nec1-sensitive ferroptoticcell deathTo define the modality of cell death, we used inducible Gpx4−/−

cells and chemical inducers for the subsequent studies. Using thepan-caspase inhibitor z-VAD-FMK (zvad) and determining caspaseactivation, we ruled out an involvement of caspases in cell deathinduced by Gpx4 loss and FINs, in contrast to parental cellstreated with staurosporine or tumour necrosis factor α (TNFα)(Supplementary Fig. 2a-c). This indicates that a non-apoptotic formof cell death is triggered in Gpx4−/− cells.

Therefore, we next addressed whether necroptosis or ferroptosis isactivated onGpx4 inactivation. Indeed, the receptor interacting kinase1 (Rip1) inhibitor necrostatin-1 (Nec1) and ferrostatin-1 (Fer1), thefirst described inhibitors of necroptosis and ferroptosis, respectively,rescued Gpx4−/− cells from cell death (Fig. 2a). As ferroptotic celldeath entails cellular iron accumulation and lipid peroxidation6, celldeath could also be inhibited by the iron chelator deferoxamine (DFO)and by αToc (Fig. 2a; ref. 11).

Likewise, depletion of the Gpx4 substrate GSH through BSO orerastin (Fig. 1a and Supplementary Fig. 2d) efficiently triggered rapidcell death (Supplementary Fig. 2e) that could be prevented by thesame inhibitors (Fig. 2b and Supplementary Fig. 2f). Furthermore,cell death induced by (1S, 3R)-RSL3 (hereafter RSL3), the firstdescribed Gpx4 inhibitor8, could also be prevented by Fer1 andNec1 (Fig. 2b), indicating that availability of cellular GSH andproper Gpx4 function tightly control ferroptosis. Consistently, allcompounds failed to induce caspase activation (SupplementaryFig. 2c), whereas Gpx4 overexpression conferred resistance to FINs(Supplementary Fig. 2g,h; ref. 14).

NATURE CELL BIOLOGY VOLUME 16 | NUMBER 12 | DECEMBER 2014 1181


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Figure 1 Inducible Gpx4 disruption causes ARF and death in mice. (a) Ascheme showing the most important steps of glutathione (GSH) biosynthesis.αToc, α-tocopherol; BSO, L-buthionine sulphoximine; GSSG, oxidizedglutathione; γGCS, γ-glutamylcysteine-synthase. (b) Inhibitors againstenzymes of arachidonic acid metabolism prevent Gpx4-deletion-induced celldeath in a dose-dependent manner. Gpx4 was disrupted in Pfa1 cells bythe addition of 1 µM TAM in the presence of increasing concentrations ofinhibitors. Cell viability was assessed by using AquaBluer 72h after knockoutinduction. Data shown represent the mean ± s.d. of n=4 wells of a 96-wellplate from a representative experiment performed independently four times.(c) Mouse survival after TAM feeding. All induced Gpx4−/− (KO) mice diedafter approximately 2 weeks of TAM feeding regardless of Alox15 expression.None of the control mice (CreERT2;Gpx4+/fl /Alox15+/+(WT/Alox15+/+),CreERT2;Gpx4+/fl /Alox15−/−(WT/Alox15−/−)) died in the period investigated.Data are percentage of live animals; mean survival of Gpx4-null miceis 13.5 days after the onset of TAM feeding (n = 8 animals forKO/Alox15−/− and WT/Alox15−/− and n= 19 animals for KO/Alox15+/+

and WT/Alox15+/+). Gehan–Breslow–Wilcoxon test: P <0.0001). (d) Overallkidney phenotype of TAM-treated CreERT2;Gpx4fl/fl animals (KO) at time

of euthanization. Left, control kidney (TAM-treated CreERT2;Gpx4+/fl , WT);right, enlarged and pale Gpx4−/− kidney. (e) Western blot of wholekidney tissue extracts showing that Gpx4 was efficiently depleted on TAMfeeding in CreERT2;Gpx4fl/fl (fl/fl,Cre), but not in control CreERT2;Gpx4+/fl

(+/fl,Cre) mice. (f) TAM-inducible CreERT2;Gpx4fl/fl mice present massivealbuminuria and unselective proteinuria compared with control mice. Eachlane represents one knockout or control animal (A, murine albumin).(g) Immunohistochemical expression analysis of Gpx4 in kidney tissuerevealed that Gpx4 was efficiently depleted on TAM treatment ofCreERT2;Gpx4fl/fl mice, which is in line with the immunoblot data. Notethe high expression of Gpx4 in tubule cells of kidney cortex, whereasglomeruli show only faint Gpx4 expression (bars top row 100 µm andbottom row 50 µm). (h) Histological analysis of kidneys of TAM-treatedCreERT2;Gpx4fl/fl animals showed widespread tubular cell death, interstitialedema and proteinaceous casts in distal tubules (bars top row 100 µm andbottom row 50 µm). (i) The number of TUNEL+ cells and mitotic cells(phosho-histone H3 staining, PH-3) is increased in kidneys of symptomaticGpx4−/− mice (bars 100 µm). Uncropped images of blots are shown inSupplementary Fig. 8.

Although ferroptosis and necroptosis are considered to be twoindependent forms of non-apoptotic cell death, we were intriguedby the protective effects of Nec1. Therefore, Rip1 was depleted inPfa1 cells by RNA-mediated interference (RNAi), but no protectionwas evident in Gpx4−/− cells (Fig. 2c). As Rip1 confers either apro-death or a pro-survival function5, we next depleted Rip3, thedownstream effector of Rip1. As Pfa1 cells express diminutive Rip3levels (Supplementary Fig. 3a), experiments were performed in L929cells showing high Rip3 expression (Fig. 2d). Rip3 expression was

efficiently decreased in L929 cells by RNAi, sufficient to preventTNFα/zvad-induced necroptosis, but not RSL3-induced cell death(Fig. 2d), or Gpx4 deletion-induced ferroptosis in Pfa1 cells (Fig. 2e).

To exclude a Rip1 contribution in ferroptosis, we treated Rip1-deficient cells with FINs, showing that Rip1−/− cells were equallysensitive to ferroptosis inducers (Fig. 2f and Supplementary Fig. 3b).Most interestingly, ferroptosis could be prevented in Rip1−/− cellsby Nec1 (Fig. 2g and Supplementary Fig. 3b), demonstrating thatNec1 has yet-unrecognized off-target effects in ferroptosis. Indeed, the



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Figure 2 The inducible Gpx4 deletion in immortalized fibroblasts causesferroptosis. (a) Cell death triggered by the inducible Gpx4 deletion in Pfa1cells can be rescued by DFO, Fer1, αToc and Nec1 in a dose-dependentmanner. Gpx4 was disrupted in Pfa1 cells as described in Fig. 1b (exceptfor DFO, which was added 24h after knockout induction, all inhibitors wereadded simultaneously along with TAM). Cell viability was assessed 72h afterknockout induction. (b) Cell death elicited in Gpx4-proficient Pfa-1 cellsby the ferroptosis inducing agents (FINs) erastin (2.5 µM), BSO (20 µM)and RSL3 (20nM) can be prevented by concomitant administration of DFO(10 µM), Fer1 (0.1 µM), αToc (1 µM) and Nec1 (20 µM), but not by the pan-caspase inhibitor Z-VAD-FMK (50 µM, zvad). Cell viability was assessed 24hthereafter. (c) Rip1 knockdown in Pfa1 cells is unable to protect against celldeath induced by Gpx4 deficiency (scrambled, scb). Cells were transfected6h after knockout induction and cell viability was assessed 66h later.(d) Rip3 knockdown attenuates TNFα/Z-VAD-FMK-induced necroptosis, butnot RSL3-induced ferroptosis in L929 cells (Scb, scrambled). Knockdownwas performed 48h before addition of the cell death stimuli. Cell viabilitywas assessed 6h after TNFα (10 ngml−1), TNFα + zvad (50 µM) or RSL3(1 µM) treatment. Data shown in c and d represent the mean ± s.d. of n=3

wells of a 12-well plate from a representative experiment performed fivetimes, P=0.05 (one-way ANOVA). (e) Rip3 knockdown in Pfa1 cells doesnot rescue cell death induced by Gpx4 knockout (scrambled, scb). Cells weretransfected 6h after knockout induction and cell viability was determined66h later. (f) Although the Rip1 inhibitor Nec1 rescued cell death triggered byFINs, Rip1-knockout (KO) cells are equally as sensitive to the Gpx4 inhibitorRSL3 as Rip1 wild-type (WT) cells; cell viability was assessed 24h aftertreatment. (g) Nec1 rescues RSL3 (1 µM)-induced cell death in both Rip1KO and WT cells in a dose-dependent manner. Cells were pre-treated withincreasing concentrations of Nec1 for 6 h and viability was assessed 24hafter treatment with FIN agents. (h) Structurally related analogues of Nec1(Nec1s, iNec) and met-Trp, an inhibitor of indoleamine 2,3-dioxygenase thatis also inhibited by Nec1, present different effects on cell death inducedby Gpx4 deletion. Rescue was performed by treating cells with increasingconcentrations of Nec1 analogues (0–100 µM), and cell viability was assessed72h after knockout induction. Data shown represent the mean ± s.d. of n=3wells (e,h) or n= 4 wells (a,b,f,g) of a 96-well plate from a representativeexperiment performed independently at least three times, P=0.05 (one-wayANOVA followed by Tukey’s multiple comparisons test).



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more specific Nec1-derivative Nec1s did not protect against Gpx4-depletion-induced cell death15 (Fig. 2h).

We next examined ultrastructural changes induced by Gpx4 inac-tivation and different cell death inducers. These studies revealed thatGpx4−/− cells exhibited swollen mitochondria, a reduced number ofcristae with a more lamellar phenotype and an increase in autophago-somes in contrast to parental Pfa1 cells (Supplementary Fig. 3c).Similar to Gpx4−/− cells, BSO-treated Pfa1 cells revealed a reducednumber of cristae with more lamellar and tubular structures, whereasautophagosomes could not be detected. Erastin-treated cells presentedmitochondria with concentric cristae. Pfa1 cells treated with TNFαshowed electron-dense whorled membranes and intermitochondrialbridges, and when treated with staurosporine, swollen mitochondriaand a loss of cristae. These data suggest that cell death induced byGpx4deletion or GSH depletion is morphologically different from that byclassical cell death inducers such as TNFα or staurosporine.

Ferroptosis is triggered by extra-mitochondrial lipidperoxidation and is characterized by the release of oxidizedlipid mediatorsLipid hydroperoxides have been described as the driving forceof ferroptosis6, although their molecular entity, mode and site ofgeneration remain unclear. Previously, we reported that Gpx4−/− cellsexhibit a yet-uncharacterized strong oxidizing component in theirlipid compartment as a measure of BODIPY 581/591 C11 oxidation16.In fact, fluorescence-activated cell sorting analysis of Gpx4−/− cellsshowed that BODIPY 581/591 C11 oxidation could be ameliorated byFer1 and DFO (Fig. 3a).

As mitochondrial lipid oxidation seems to be a critical step inthe execution of some forms of cell death17, we further addressedwhether a ‘lethal lipid signal’ is generated in mitochondria orextra-mitochondrial compartments. Therefore, we comparativelymonitored the oxidation of mito- versus non-targeted BODIPY581/591 C11 in Pfa1 cells treated with RSL3 (Fig. 3b). Interestingly,the non-targeted redox sensor presented a faster oxidation kineticscompared with the mitochondria-targeted one. Furthermore, thisoxidation preceded cell lysis (Fig. 3c), indicating that the initial lipidperoxidation step occurs outside the mitochondrial matrix. Moreover,ultrastructural analysis demonstrated that ferroptosis involves outermitochondrial membrane (OMM) rupture (Fig. 3d), possibly markingthe point of no-return in ferroptosis. Surprisingly, cells treatedwith thelipophilic antioxidant decylubiquinone and its mitochondria-targetedchemical analogue, mitoquinone mesylate (MitoQ), required 100-fold higher MitoQ concentrations to prevent ferroptosis (Fig. 3e),supported by de-localizing the targeted antioxidant with FCCP(Supplementary Fig. 3d)18. This implies that ferroptosis-inducedOMM disruption proceeds through a mechanism not amenable tomitochondria-targeted antioxidant therapies.

Reportedly, enriching membranes with arachidonic acidaccelerated the cell death process on Gpx4 deletion11. As such,we determined arachidonic acid metabolites released into the cellculturemedium on triggering ferroptosis.We now show that the levelsof 5-hydroxyeicosatetraenoic acid (HETE), 11-HETE and 15-HETEwere increased in the medium of Gpx4−/− cells 48 h after TAMtreatment (Fig. 4a), which could be abrogated by zileuton, MK886,PD146176, MJ33, Fer1 and Nec1 (Fig. 4b). In contrast, extracellular

12-HETE levels remained unaffected in response toGpx4 loss, arguingagainst an unspecific lipid oxidation (Fig. 4a). MEFs treated with FINsalso presented a similar HETE signature (Fig. 4c), demonstratingthat lipid mediators are released during ferroptosis. Accordingly,the cell lines described in Supplementary Fig. 1a,b demonstratethat Alox15 deficiency with Alox5 knockdown presented a slowerrelease of lipid oxidation products than parental cells (SupplementaryFig. 4a,b), thus probably explaining their increased resistance toFINs (Supplementary Fig. 4c). Accordingly, treatment of Gpx4−/−

cells with 5-, 12- and 15-hydroperoxyeicosatetraenoic acid (HpETEs)accelerated the ferroptotic process, whereas other metabolites,including HETEs and oxoeicosatetraenoic acid, did not influence celldeath (Fig. 4d). Equimolar concentrations of H2O2 treatment did notphenocopy the effects of HpETEs, ruling out a general peroxide effect.

Despite the strongly reduced protection of mitochondria-targetedantioxidants (Fig. 3e), it is evident that morphological (Fig. 3d) andchemical (Fig. 4) changes occur in mitochondria and contribute toferroptosis. Lipid transport proteins, particularly sterol carrier protein2 (SCP-2), disseminate oxidative stress between compartments19 bypreferentially trafficking peroxidized lipids to mitochondria20. Infact, SCP-2 inhibitors21 showed anti-ferroptotic activity (Fig. 4e),which was substantiated by knocking down SCP-2 in Gpx4−/− cells(Supplementary Fig. 4d). Yet, this effect was transient as it only sloweddown cell death kinetics (Supplementary Fig. 4e), implying that othertranslocases may work in parallel with SCP-2.

Oxi-lipidomics analysis reveals a complex lipid oxidationsignature of Gpx4-null kidneysIn an untargeted approach, we set out to directly assess lipidoxidation products in Gpx4-null kidneys using lipid extraction andLC–MS analysis22. These investigations were deemed necessary tobypass the use of fluorescence-based redox sensors, whose mode-of-actions are poorly characterized and may lead to potentiallymisleading conclusions.

Using oxidative lipidomics, we established that inGpx4−/− kidneysphosphatidylcholine (PC) and phosphatidylethanolamine (PE), aswell as the mitochondria-specific phospholipid, cardiolipin (CL),underwent substantial oxidative modifications. To assign specific acylchain combinations for individual phospholipid molecular species,including PC, we performed MS/MS analysis using the negativeionizationmode. For example, theMS/MSpattern of the PCmolecularion withm/z 816.556 [M–H+CH3COOH]− clearly demonstrates thepresence of carboxylated ions with m/z 255.232 and m/z 279.232corresponding to 16:0 and 18:2 fatty acid residues. The potentiallyconfounding carboxylated daughter ions—withm/z 253.232 andm/z281.232 corresponding to 16:1 and 18:1—were not detected in theMS/MS spectrum. We have presented MS/MS spectra for PC (m/z816.556), PE (766.539) and CL (1,447.961) in Supplementary Fig. 5.

As Gpx4 catalyses the reduction of double-oxygenatedphospholipids into mono-oxygenated phospholipids, we performedLC–MS assessment of these respective species in kidneys fromcontrol versus Gpx4−/− animals on day 6 when no manifestationsof renal failure were detectable and on day 10 with an overt renalfailure phenotype (Fig. 5). To verify the nature of double oxygenatedspecies, we treated lipid extracts with a mild reducing agent,triphenylphosphine, converting hydroperoxy (OOH) to the respective



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0 2 4 6h

0 min 20 min 1 h 3 h

Per

cent

age

of c

ell v

iab

ility

0

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0

30

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120

0

30

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0

30

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10–10 10–810–9 10–7 10–6 10–5

(M)

10–10 10–810–9 10–7 10–6 10–5

(M)

10–10 10–810–9 10–7 10–6 10–5

(M)

10–10 10–810–9 10–7 10–6 10–5

(M)

Gpx4 KO Erastin RSL3 BSO

DecylQMitoQ

DecylQMitoQ

DecylQMitoQ

DecylQMitoQ

Figure 3 Lipid peroxidation outside the mitochondrial matrix triggersferroptosis in MEFs. (a) Inducible knockout of Gpx4 leads to increasedlipid peroxidation that can be blunted by Fer1 and DFO. Lipid peroxidationwas measured 48h after knockout induction using the redox-sensitive dyeBODIPY 581/591 C11. (b) RSL3 causes rapid lipid peroxidation outsidethe mitochondrial matrix as determined by BODIPY 581/591 C11 versusmitochondrial-targeted BODIPY 581/591 C11 staining; oxidation of theprobe was assessed at the indicated time points. (c) Time-dependent increasein LDH release in Pfa1 cells treated with RSL3 (100nM); supernatantswere collected at the indicated time points and assayed for LDH activity.Data shown represent the mean ± s.d. of n=3 wells of a 12-well platefrom an experiment performed independently five times, P=0.05 (one-way

ANOVA). (d) Electron micrographs showing a time-dependent OMM rupture(yellow arrows) on ferroptosis induction using RSL3 (50nM; scale bars 2 µmtop row, 200nm bottom row). (e) The mitochondrial targeted antioxidantMitoQ is by far less efficient in protecting cells against ferroptotic celldeath, elicited by Gpx4 knockout (KO), erastin, RSL3 or BSO, comparedwith the membrane-targeted antioxidant DecylQ. Ferroptosis was induced byGpx4 knockout in Pfa1 cells by 1 µM TAM or by treatment of non-inducedPfa1 cells with FINs in the presence of increasing concentrations of theinhibitors; cell viability was determined 72h and 24h later, respectively.Data shown represent the mean ± s.d. of n=4 wells of a 96-well plate froma representative experiment performed independently at least three times,P=0.05 (one-way ANOVA).

hydroxy (OH) species (Supplementary Fig. 5d). As illustrated inFig. 5 the di-oxygenated species of phospholipids in Gpx4 mice wererepresented by OOH-species and their level was significantly higherin PC (Fig. 5a) and PE (Fig. 5b) on day 10 after Gpx4 depletion.

Next, we focused on examining OOH-derivatives of amitochondria-specific phospholipid, CL, that is recognized asa preferred Gpx4 substrate23. A significantly higher content ofCL–OOH species was detected in Gpx4−/− kidneys on day 6

compared with day 10 (Fig. 5c). In addition, for both of these timepoints the level of CL–OOH was significantly higher compared withcontrol mice. Moreover, we found that on both days, Gpx4-nullmice accumulated more mono-lyso-CL (mCL) (Fig. 5c) as well ashydroperoxy-molecular species of free fatty acids, including linoleic,arachidonic and docosahexaenoic acid (Fig. 5e). Significant changesin accumulation of lyso-PC (LPC) and lyso-PE (LPE) in kidneys ofGpx4−/− mice were detectable only on day 10 (Fig. 5d).



ART ICLES

∗

∗ ∗

0

0.4

0.8

WTKO

∗

nd nd

∗∗

∗

0

0.2

12-H

ETE

(pg

ml–1

)

∗ ∗∗

∗

ControlZilTAMTAM + Zil

∗

∗

∗ ∗∗

∗

∗

∗

∗

∗

∗

∗

∗

∗

∗

∗

∗

∗

∗

∗

20406080

100120

5-H(p)ETE 12-H(p)ETE

15-H(p)ETE

WTKO

∗

∗∗

∗∗

SCPI 20

30

60

90

120 00.1110

μM

20406080

100120

20406080

100120

20406080

100120

Ethan

ol

HETE

HpETE

OxoHETE

Ethan

ol

HETE

HpETE

OxoHETE

Ethan

ol

HETE

HpETE

OxoHETE

Ethan

olH 2

O 2

Per

cent

age

ofce

ll vi

abili

tyP

erce

ntag

e of

cell

viab

ility

Per

cent

age

ofce

ll vi

abili

tyP

erce

ntag

e of

cell

viab

ility P

erce

ntag

e of

cel

l via

bili

ty

SCPI 3

SCPI 4

SCPI 5

0

0.6

1.2

1.8

2.4

5-HETE

11-H

ETE

12-H

ETE

15-H

ETE

5-HETE

11-H

ETE

12-H

ETE

15-H

ETE

HE

TEs

(pg

ml–1

)H

ETE

s (p

g m

l–1)

0

0.6

1.2

1.8

2.4ControlMK866TAMTAM + MK866

5-HETE

11-H

ETE

12-H

ETE

15-H

ETE

5-HETE

11-H

ETE

12-H

ETE

15-H

ETE

HE

TEs

(pg

ml–1

)H

ETE

s (p

g m

l–1)

0

0.6

1.2

1.8

2.4ControlPD146176TAMTAM + PD146176

ControlMJ33TAMTAM + MJ33

ControlFer1TAMTAM + Fer1

ControlNec1TAMTAM + Nec1

0

0.6

1.2

1.8

2.4

5-HETE

11-H

ETE

12-H

ETE

15-H

ETE

5-HETE

11-H

ETE

12-H

ETE

15-H

ETE

HE

TEs

(pg

ml–1

)H

ETE

s (p

g m

l–1)

0

0.6

1.2

1.8

2.4

0.4

0.6

0.8

0

0.2

15-H

ETE

(pg

ml–1

)

0.4

0.6

0.8

011-H

ETE

(pg

ml–1

)

0.1

0.2

0.3

00.1

5-H

ETE

(pg

ml–1

)

0.20.30.40.5

Contro

lTA

MEra

stinRSL3

TNFα

Staur

o

Contro

lTA

MEra

stinRSL3

TNFα

Staur

o

Contro

lTA

MEra

stinRSL3

TNFα

Staur

o

Contro

lTA

MEra

stinRSL3

TNFα

Staur

o

20 40 60 80h

20 40 60 80h

20 40 60 80h

20 40 60 80h

0

0.4

0.8

0

0.12

0.24

5-H

ETE

(pg

ml–1

)0

0.2

0.4

11-H

ETE

(pg

ml–1

)

12-H

ETE

(pg

ml–1

)

15-H

ETE

(pg

ml–1

)

a c

b

d e

0

0.6

1.2

1.8

2.4

Figure 4 Ferroptosis is characterized by the release of lipid mediators.(a) Quantification of a time-dependent release of arachidonic acid metabolites(5-HETE, 11-HETE, 12-HETE and 15-HETE) in the cell culture mediaafter inducible Gpx4 deletion (KO) using LC–MS/MS analysis. (b) Impact ofsmall-molecule inhibitors on HETE release on Gpx4 deletion. Quantificationof released arachidonic acid metabolites was performed by LC–MS/MS.Cells were treated with zileuton (10 µM), MK866 (50 µM), PD146176(1 µM), MJ33 (10 µM) and Fer1 (0.2 µM) at the time of knockout induction.Analysis was performed in conditioned medium collected 72h after Gpx4deletion. (c) Quantification of arachidonic acid metabolites released into themedium 24h after cell death induction by erastin (2.5 µM), RSL3 (0.75 µM),TNFα (10 ngml−1) and staurosporine (Stauro; 0.2 µM) using HPLC–MS/MSanalysis. In the Gpx4−/− cells, HETE levels were determined 72h after TAMtreatment. Data shown represent the mean ± s.d. of n=3 wells of a 6-wellplate from an experiment performed independently three times (a,c) or one

time (b). ∗P=0.05, ∗∗P=0.01 (one-way ANOVA, followed by Tukey’s multiplecomparisons test (a) or Dunnett’s multiple comparison test (b,c)). (d) Impactof arachidonic acid metabolites on the cell death process elicited by Gpx4loss. HETEs, HpETEs and OxoHETEs (1 µM) were added to WT or KO Pfa1cells for Gpx4. Compounds were added 36h after knockout induction andcell viability was measured 12h thereafter. H2O2 was used at equimolarconcentrations. Data shown represent the mean ± s.d. from n= 4 wells(HpETEs) or n=6 wells (H2O2) of a 96-well plate from a representativeexperiment performed three times, ∗P=0.05, ∗∗P=0.01, one-way ANOVAfollowed by Tukey’s multiple comparisons test. (e) Sterol carrier protein-2(SCP) inhibitors (SCPI) rescue Gpx4−/− cells from ferroptosis in a dose-dependent manner. Inhibitors were added concomitantly to TAM and cellviability was determined 72h thereafter. Data shown represent the mean± s.d. of n=6 wells of a 96-well plate from a representative experimentperformed independently two times, P=0.05 (one-way ANOVA).



ART ICLES

02468

1012

PE

–OO

H (p

mol

per

nm

ol P

E)

(C16:0/C20:3–OOH)(C18:1/C18:2–OOH)

(C16:0/C20:4–OOH)(C18:2/C18:2–OOH)

0

20

40

60

80

100 ∗∗

∗

(18:0/20:4–OOH)

0

40

80

120

6 days 10 days 6 days 10 days 6 days 10 dayspm

ol p

er n

mol

tot

al P

Ls

18:2–OOH 20:4–OOH 22:6–OOH

∗

∗

∗ ∗

∗

e

b

CreERT2;Gpx4+/fl

CreERT2;Gpx4fl/fl

0

2

4

6

8

mC

L (p

mol

per

nm

ol P

Ls)

1,161.5

1,185.5

1,163.5

1,209.5

1,187.5

1,213.51,233.5

1,120 1,160 1,200 1,240 m/z

1,187.51,185.5

1,213.51,209.5

1,233.51,163.5

∗mCL

6 days 10 days

m/z

0

5

10

15

20

25

6 days 10 days 6 days 10 days

CL–

OO

H (p

mol

per

nm

ol C

L)

∗

∗

∗

∗

(18:2)3/(18:2–OOH)1 (18:1)1/(18:2)2/(18:2–OOH)1

OO

HO

O

NH2

P OHO

OOH

OO

700 720 740 760 780 800 m/z

722.513750.545

790.538764.524

774.542

(18:0)(20:4)766.539

(C16:0)(C20:4)(C18:2)(C18:2)

738.508

(C16:0)(C20:3)(C18:1)(C18:2)740.524

c

(O-16:0/22:6–OOH)(18:0/20:4–OOH)(16:0/18:2–OOH)

∗

∗

∗

a

OO

HOO

N+

P O–O

OO

OOH

760 780 800 820 840 860 880 900 920 m/z

792.576

892.607922.579

(16:0)(18:2)816.576

(18:2)(18:2)840.576

(18:0)(18:2)844.607

(18:1)(20:4)866.588

(16:0)(22:6)864.576

(O-16:0)(22:6)850.596

(18:0)(20:4)868.607

PO OOH

OO

H

H

HOO

PO OHOO

OO

OO

HO

OOH

O

1,400 1,420 1,440 1,460 1,480 1,500 1,520 m/z

1,475.992

1,427.995 1,497.9751,403.994

1,523.992

(18:2)41,447.961

(18:1)1(18:2)31,449.977

d

0

1

2

3

4

02468

10

540.3

568.3

566.3

568.3

530 540 550 560 m/z

540.3

566.3

450 470 m/z

480.3

452.3 478.3

LPC

∗

452.3

480.3

478.3

m/z

LPE

LPE

(pm

ol p

er n

mol

PLs

)

wt/fl fl/fl

LPC

(pm

ol p

er n

mol

PLs

)

fl/flwt/fl

∗ m/z

0

4

8

12

16

20

24

PC

–OO

H (p

mol

per

nm

ol P

C)

Figure 5 LC–MS characterization of phospholipids and their oxidationproducts in kidneys of Gpx4-null and wild-type mice. (a) Mass spectrumof PC obtained from kidneys of Gpx4-null mice (left panel) and thelevels of hydroperoxy-PC species in Gpx4-null (CreERT2;Gpx4fl/fl ) and wild-type (CreERT2;Gpx4+/fl ) mice (right panel). Hydroperoxy-PC species (m/z848.575, 872.575, 876.606, 882.595, 896.575, 898.587 and 900.606)originated from PC species with m/z 816.576 (16:0/18:2), 840.576(18:2/18:2), 844.607 (18:0/18:2), 850.596 (O-16:0/22:6), 864.576(16:0/22:6), 866.588 (18:1/20:4) and 868.607 (18:0/20:4). Inset:structural formula of PC–OOH—(16:0/18:2–OOH). (b) Mass spectrum of PEobtained from kidney of Gpx4−/− mice and the content of hydroperoxy-PEspecies in Gpx4-null mice (right panel). Oxygenated species (m/z 770.507and 772.522 and m/z 798.538) originated from major PE molecularspecies with m/z 738.508 (18:2/18:2 or 16:0/20:4), 740.524 (18:1/18:2or 16:0/20:3) and 766.539 (18:0/20:4). Inset: structural formulaof PE–OOH containing hydroperoxy-arachidonic acid—(18:0/20:4–OOH).(c) Mass spectrum of CL obtained from kidney of Gpx4−/− mice (leftpanel) and levels of hydroperoxy-CLs (m/z 1,479.932 and m/z 1,481.975)originating from species with m/z 1,447.961 (18:2)4 and 1,449.97718:1/(18:2)3, respectively (right panel). Inset: structural formula of CL–OOH

containing hydroperoxy-linoleic acid—(18:2)3/18:2–OOH. (d) Mass spectraand levels of lysophospholipids in samples from mouse kidneys. LPC,lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; mCL, mono-lysocardiolipin. (e) Content of hydroperoxy-derivatives of linoleic (18:2–OOH),arachidonic (20:4–OOH) and docosahexaenoic (22:6–OOH) acids in samplesfrom mouse kidneys. Mass spectra of PC, PE and CL were acquired usinga QExactive orbitrap mass spectrometer (ThermoFisher Scientific). Theresolution was set up at 140,000 corresponding to 5 ppm deviation in m/zmeasurement. Mass spectra of LPC, LPE and mCL were acquired by anLXQ ion trap mass spectrometer (ThermoFisher Scientific). Consequently,m/z values are presented to three and one decimal places, respectively.PC molecular ions were detected in a negative ionization mode asacetate adducts [M–H+CH3COOH]−. LPC molecular ions were detectedin a negative ionization mode as formate adducts [M–H+HCOOH]−. PEand CL as well as LPE and mCL molecular ions were detected ina negative ionization mode as singly charged ions [M–H]−. Data forhydroperoxy-species are presented as picomoles per nanomole of the parentalnon-oxidized species. Data shown represent the mean ± s.d. of n= 4animals of a representative experiment performed independently two times,P=0.05 (one-way ANOVA).



ART ICLES

As oxidized PLs are preferred substrates for cytosolic andmitochondrial aiPLA2 (ref. 24), which can be inhibited by MJ33(Fig. 1b), we speculate that the remarkable accumulation of mCLmight account for the disrupted morphological changes observedon Gpx4 disruption (Fig. 3d). Moreover, because the mito-targetedredox sensors and antioxidant MitoQ localize to the matrix and theinner leaflet of the mitochondrial inner membrane and because ofthe finding that Gpx4 resides in the mitochondrial inner membranespace25, we propose a yet-unrecognized oxidation event that mayoccur at specific submitochondrial locations outside the matrix,which is supported by ultrastructural analysis of cells treated withRSL3 (Fig. 3d). In sum, these data demonstrate that distinctive lipidhydroperoxides accumulate in tissues that are centrally involved inferroptosis induced by Gpx4 deletion.

A spiroquinoxalinamine derivative inhibits ferroptosis inthe kidneyConsidering the apparently high modularity of ferroptosis, we soughtto identify small-molecule inhibitors with potent anti-ferroptoticproperties. We took advantage of the correlation between the in vitroand in vivo phenotype of Gpx4−/− cells and mice, which permittedus to immediately test the in vivo efficacy of screened molecules.We conducted a screening campaign, testing over 40,000 drug-likesmall molecules in TAM-inducible Gpx4−/− MEFs. This resulted inthe discovery of a yet-unrecognized class of spiroquinoxalinaminederivatives, including Liproxstatin-1 (Fig. 6a), which is able to inhibitferroptosis in the low nanomolar range (Fig. 6b). Liproxstatin-1 pre-vented BODIPY 581/591 C11 oxidation in Gpx4−/− cells (Fig. 6c).Moreover, Liproxstatin-1 did not interfere with other classical types ofcell death, such as TNFα-induced apoptosis andH2O2-induced necro-sis (Fig. 6d), and in the bona fide L929 model of TNFα/zvad-inducednecroptosis (Fig. 6e). In addition, testing Liproxstatin-1 against a panelof different cell death inducers unrelated to ferroptosis confirmedthe specific anti-ferroptotic activity of Liproxstatin-1 (SupplementaryFig. 6). In stark contrast, it showed remarkably potent and specificanti-ferroptotic activity against FIN-triggered cell death (Fig. 6e).

To establish an initial structure–activity relationship study (SAR),we obtained a handful of commercially available analogues ofLiproxstatin-1. The compound structures and corresponding IC50

values are depicted in Fig. 6f. In addition, we assessed importantADME (absorption, distribution, metabolism and excretion)parameters (Table 1), indicating a very promising pharmacokineticprofile for Liproxstatin-1. In summary, the initial SAR resultsprovide the rationale for further improvement of Liproxstatin-1 bymedicinal chemistry.

Liproxstatin-1 suppresses ferroptosis in human cells, Gpx4−/−

kidney and in an ischaemia/reperfusion-induced tissueinjury modelTo assess the relevance of these findings in humanbiology, primary hu-man renal proximal tubule epithelial cells (HRPTEpiCs) were treatedwith RSL3. As shown in Fig. 7a, RSL3 efficiently triggered cell deathin HRPTEpiCs, whereas the inactive analogue ((1R,3R)-RSL3) didnot. The specificity of RSL3 was confirmed by Gpx4 overexpression(Supplementary Fig. 7a,d). Supporting the involvement of ferroptosis,treatment with Liproxstatin-1 was able to protect HRPTEpiCs from

Table 1 ADME-Tox profiling of Liproxstatin-1.

Cytotoxicity in HepG2 cells TC50 (µM) 6

hERG patch clamp assay IC50 (µM) >25

CYP450 inhibition 2C9 IC50 (µM) >25

2D6 IC50 (µM) =4.1

3A4 IC50 (µM) >25

1A IC50 (µM) >25

2C19 IC50 (µM) >25

Mouse PK (i.v.) 1mg kg−1 t1/2 (h) 4.6

V (ml) 849

C (µgml−1) 0.4

Total Cl (ml h−1) 128

Total Cl (mlmin−1 kg−1) 66

MRTINF (h) 4.9

Vss (ml) 629

Mouse PK (p.o.) 5mg kg−1 Cmax (µgml−1) 0.08

Tmax (h) 2

Bioavailability (%) 52An initial ADME-Tox profile for Liproxstatin-1 is provided, including cytotoxicity inHepG2 cells and inhibition of hERG and cytochrome P450 (CYP450) enzymes, as wellas basic pharmacokinetic parameters. hERG, ether-à-go-go-related gene; ADME-Tox,absorption, distribution, metabolism and excretion—toxicity; PK, pharmacokinetics;Cl, clearance; MRTINF, mean residence time extrapolated to infinity.

RSL3-induced cell death (Fig. 7a). Similar findings were obtained inthe immortalized human renal proximal tubule epithelial cell line,HK-2 (Supplementary Fig. 7b). Next, we knocked down Gpx4 inHK-2 cells using a pool of siRNAs, revealing a small yet significantdecrease in cell viability sensitive to αToc treatment (SupplementaryFig. 7c). Inducing cell death through Gpx4 knockdown, however,turned out to be challenging for the high expression levels of Gpx4 inkidney tubular epithelial cells (Supplementary Fig. 7d). Nonetheless,the Gpx4 knockdown rendered cells more sensitive to ferroptosis-inducing agents (Supplementary Fig. 7e), indicating a Gpx4-regulatedferroptotic machinery in human proximal tubular epithelial cells.Moreover, RSL3-induced BODIPY 581/591 C11 oxidation could beblocked by Liproxstatin-1 (Fig. 7b), demonstrating that Liproxstatin-1prevents ferroptotic cell death also in humans.

Next, we assessed the in vivo potential of Liproxstatin-1 to preventthe consequences of inducible Gpx4 disruption in animals. OnTAM treatment of CreERT2;Gpx4fl/fl mice, mice were injected dailywith Liproxstatin-1 intraperitoneally (i.p.) until the mice showedsigns of acute renal failure (ARF), at which point they wereeuthanized (Fig. 7c). Notably, Liproxstatin-1 remarkably extendedsurvival compared with the vehicle-treated group. TUNEL stainingat day 9 after TAM treatment showed a strongly reduced number ofTUNEL+ cells in Liproxstatin-1 compared with the vehicle-treatedgroup (Fig. 7d), suggesting that Liproxstatin-1 delays ferroptosis intubular cells. The discrepancy between death of mice due to ARF inFig. 1c and vehicle-treated animals in Fig. 7c is explained by the modeof TAM administration, feeding versus i.p. injection.

As an independent proof-of-concept, we analysed the invivo efficacy of Liproxstatin-1 in a bona fide model of hepaticischaemia/reperfusion injury, providing evidence that Liproxstatin-1



ART ICLES

N

HN

NH

NH

Cl

N

HN

NH

Cl

N

HN

NH

Cl

N

HN

NH

N

HN NH

NH

N

HN NH

NH

ClN

HN NH

NH

Cl

Liproxstatin-1IC50 = 22 nM

IC50 = 26 nM

IC50 = 25–50 nM

IC50 = 269 nM

IC50 = 171 nM

IC50 > 1,000 nM

a b c

0

20

40

60

80

100

Per

cent

age

of M

ax

Gpx4 KO + Liproxstatin-1Gpx4 KOGpx4 WT

0 102 103 104 105

Alexa-488

10–9 10–710–8 10–6 10–5 10–40

25

50

75

100

125

Per

cent

age

of c

ell v

iab

ility

(M)

Gpx4 WTGpx4 KO + Nec-1Gpx4 KO + Liproxstatin-1Gpx4 KO + Fer1

Contro

l

DMSO

Nec1

Nec1s

Lipro

xsta

tin-1

Fer1

Per

cent

age

of c

ell v

iab

ility

0

30

60

90

120L929

TNFα + zvad

e fd

Per

cent

age

of c

ell v

iab

ility

0

30

60

90

120

3000 100 200

BSO (10 μM) Erastin (1 μM)

RSL3 (0.5 μM) Stauro (0.2 μM)

H2O2 (200 nM)

Liproxstatin-1 (nM)

Figure 6 Identification and characterization of ferroptosis inhibitors with invivo efficacy. (a) Chemical structure of Liproxstatin-1. (b) Dose-dependentrescue of ferroptosis by Liproxstatin-1 in Gxp4−/− cells. On Gpx4 disruptionin Pfa1 cells by TAM in the presence of Liproxstatin-1, cell viability was deter-mined 72h after knockout induction. (c) Liproxstatin-1 (50 nM) completelyprevents lipid peroxidation in Gpx4−/− cells. Lipid peroxidation was assessed48h after knockout induction using the redox-sensitive dye BODIPY 581/591C11. (d) Specificity of Liproxstatin-1 towards ferroptosis-inducing triggers.Liproxstatin-1 (200nM) protects against FINs, such as BSO (10 µM), erastin(1 µM) and RSL3 (0.5 µM), in a dose-dependent manner, whereas it failsto rescue cell death induced by staurosporine (Stauro; 0.2 µM) and H2O2

(200 µM); cell viability was assessed 24h after treatment. (e) Liproxstatin-1does not prevent necroptosis. Two hours before triggering necroptosis, L929cells were treated with Nec1 (10 µM), Nec1s (10 µM), Liproxstatin-1 (1 µM)and Fer1 (1 µM). Necroptosis was induced in L929 cells by a combination ofTNFα (5 ngml−1) and Z-VAD-FMK (50 µM) and cell viability was determined8h thereafter using AquaBluer. Data shown represent the mean ± s.d. ofn=4 wells (b,e) or n=3 wells (d) of a 96-well plate from a representativeexperiment performed independently at least four times. (f) An initial SARanalysis including IC50 values of Liproxstatin-1 derivatives is shown. IC50

values depicted were calculated from experiments performed with inducibleGpx4−/− cells in 1 well of a 96-well plate, pooled from n=4.

mitigated tissue injury in ischaemia/reperfusion-induced liver injury(Fig. 7e). Hence, these data implicate ferroptosis as a contributor inischaemia/reperfusion-induced tissue injury and hold great promisefor the development of therapeutics to treat related pathologies.

DISCUSSIONGpx4 is one of the most important antioxidant enzymes inmammals11,26,27. This vital role is mainly credited to its uniquefunction as the only known enzyme able to reduce phospholipidhydroperoxides28. After having shown that loss of Alox15 alonecannot rescue ARF and cell death induced by Gpx4 inactivation,we provide here a more intricate picture where other lipoxygenaseisoforms may compensate for Alox15 deficiency. Although sources ofreactive oxygen species other than lipoxygenases, such as NADPHoxidases, have been implicated, their contribution seems to be cell

type dependent, whereas lipid-oxidizing enzymes may confer a moregeneral impact6,7. In addition, we introduce the release of fattyacids through aiPLA2 and its trafficking possibly through SCP-2 asdownstream events involved in ferroptosis execution.

A direct measurement of PL oxidation products by LC–MS analysisin vivo showed higher levels of di-oxygenated species of PC, PE andCLin the kidneys ofGpx4−/−mice versus control animals, indicating thatnot only extra-mitochondrial compartments but also mitochondriawere involved in the deregulated lipid peroxidation caused by Gpx4deficiency. Yet, rescue of Gpx4−/− cells with mitochondria-targetedantioxidants was far less efficient than with untargeted ones, arguingthat the priming lethal lipid signal must be generated outside themitochondrial matrix.

In accordance with a role for ferroptosis in vivo, we nowdemonstrate that proper kidney function depends on a functional



ART ICLES

∗

a b c

d e

Per

cent

age

of c

ell v

iab

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Figure 7 Ferroptosis in human cells and in murine disease models can betargeted by Liproxstatin-1. (a) HRPTEpiCs are susceptible to Gpx4-inhibition-induced ferroptosis, which can be prevented by Liproxstatin-1 (100nM).Dose-dependent killing of HRPTEpiCs by active (1S,3R)-RSL3 in contrastto inactive (1R,3R)-RSL3. Viability was assessed 24h after treatment usingAquaBluer. Data shown represent the mean ± s.d. of n=3 wells of a 96-well plate from a representative experiment performed independently threetimes. (b) Liproxstatin-1 prevents RSL3 (0.2 µM)-induced lipid oxidation inHRPTEpiCs. Lipid peroxidation was assessed 24h after knockout inductionusing the redox-sensitive dye BODIPY 581/591 C11. A representativeexperiment is shown performed independently four times. (c) Liproxstatin-1 retards ARF and death of mice induced by Gpx4 deletion; mediansurvival was calculated to be 11 days for vehicle-treated (n= 12) and

14 days for Liproxstatin-1-treated mice (n=13), Gehan–Breslow–Wilcoxontest: P <0.0001. Representative experiment shown was performed twotimes. Mice were injected daily with Liproxstatin-1 (10mg kg−1, i.p.) duringthe course of the experiment. (d) Quantification of TUNEL+ cells inkidneys of vehicle- and Liproxstatin-1-treated animals at 9 days afterTAM administration. Data shown represent the mean ± s.d. of n = 4comparable anatomical sections from a representative experiment performedtwo times (scale bars 50 µm). (e) The extent of tissue injury on transientischaemia/reperfusion in liver of C57BL/6J mice can be ameliorated bythe ferroptosis inhibitor Liproxstatin-1 as measured by AST/ALT (n= 17)for vehicle and for Liproxstatin-1 each) and by determining the necroticarea (n=5). Data represent the mean ± s.e.m.; ∗P=0.05 or ∗∗∗P=0.001(one-way ANOVA) followed by Dunnett’s post-test.

Gpx4 protein, as the inducible Gpx4 depletion leads to massivecell death of renal tubular epithelia. Interestingly, a recent reportusing inducible ferritin heavy chain (FtH )-knockout mice in kidneypresented a similar phenotype on different challenges that isdependent on a non-haem iron mechanism29, thus highlighting theimportance of restraining lipid peroxides and iron in tubular cellsas these are expected to fuel the ferroptotic pathway and culminatein ARF. It is worth mentioning that in the FtH -null mouse, nodamagewas observed under basal conditions, indicating that pure ironoverload is not sufficient to elicit ferroptosis. However, the primingeffect would be an increase in lipid hydroperoxides to an extent thatiron would work only as a catalyst. Nonetheless, we point out thatalthough the iron chelator DFO prevented cell death induced by Gpx4loss in vitro, the iron dependence of renal cell death in the inducibleGpx4−/− model remains to be formally shown in vivo.

As we provide evidence that ferroptosis plays an important rolein ischaemia/reperfusion-induced tissue damage, targeting other non-apoptotic pathways might help pave the way towards improvedprotection against ischaemia30. It is therefore intriguing to considerhow ferroptosis is functionally intertwined with other types ofcell death4. We also highlight that because Nec1 has been usedpreviously to define necroptotic cell death, but can also protectagainst ferroptosis through an unknown target, many earlier studiesmay now be suspected to encompass ferroptotic cell death. Hence,the finding that the ferroptotic signalling cascade is amenable to invivo pharmacological intervention (Fig. 7) opens new possibilitiesfor redox-targeted therapies. Last, this study provides the rationalefor future investigations concerning the role of how lipid-derivedelectrophiles translate their message to redox-sensitive proteinstriggering cellular demise31. �



ART ICLES

METHODSMethods and any associated references are available in the onlineversion of the paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSWe are grateful to D. Green for his valuable comments and critical reading ofthe manuscript. We would also like to thank A. Berns, The Netherlands CancerInstitute, Amsterdam, for providing the ROSA26-CreERT2 mouse line, J. Silke (TheWalter and Eliza Hall Institute, Melbourne, Australia) for the Rip1-knockout cellsand M. P. Murphy (Medical Research Council, London, UK) for provision of MitoQandMito-BODIPY.We aremost grateful to L. Pichl for excellent technical assistance.This work was in part supported by the Deutsche Forschungsgemeinschaft (DFG)CO 291/2-3 toM.C., a fellowship fromAlexander vonHumboldt-Stiftung to J.P.F.A.,a fellowship from the Japan Society for the Promotion of Science (JSPS) to S.K., theHuman Frontier Science Program (HFSP) RGP0013 to M.C. and V.E.K., HL114453(NIH) to V.E.K., and by the m4 Award (Bavarian Ministry of Economic Affairs) toM.C. and J.A.S.

AUTHOR CONTRIBUTIONSJ.P.F.A. and M.C. conceived the study. Electron microscopy studies were carried outand analysed byM.A. andA.W. Eicosanoid release and data analysis were performedby V.J.H. and V.B.O. Oxi-lipidomics analysis and interpretation was performedby Y.Y.T., V.A.T. and V.E.K. Optimized RSL3 synthesis was conceived and carriedout by S.B.T. and B.R.S. Liver ischaemia/reperfusion experiments were performedand analysed by E.E. and E.K.G. Gpx4/Alox15 double-knockout generation andhistopathological analysis was conceived and performed by G.W.B., N.H., H.B., F.N.,I.E. and R.W. Small-molecule screen was conceived and analysed by B.P., J.A.S. andM.C. Experiments were performed by J.P.F.A., M.S., N.H., D.B., S.K., T.S., H.F., O.Y.andM.H. Evaluation and interpretation of the in vitro datawas performed by J.P.F.A.,O.R. and M.C. Paper was written by J.P.F.A., V.E.K. and M.C. All authors read andcommented on the paper.

COMPETING FINANCIAL INTERESTSThe authors declare that a patent application has been filed for some of thecompounds described in this work.

Published online at www.nature.com/doifinder/10.1038/ncb3064Reprints and permissions information is available online at www.nature.com/reprints

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17. Kagan, V. E. et al. Cytochrome c acts as a cardiolipin oxygenase required for releaseof proapoptotic factors. Nat. Chem. Biol. 1, 223–232 (2005).

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20. Vila, A., Levchenko, V. V., Korytowski, W. & Girotti, A. W. Sterol carrier protein-2-facilitated intermembrane transfer of cholesterol- and phospholipid-derivedhydroperoxides. Biochemistry 43, 12592–12605 (2004).

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22. Tyurina, Y. Y. et al. Characterization of cardiolipins and their oxidation products byLC-MS analysis. Chem. Phys. Lipids 179, 3–10 (2014).

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http://www.nature.com/doifinder/10.1038/ncb3064





METHODS DOI: 10.1038/ncb3064

METHODSChemicals. All chemicals were purchased from Sigma-Aldrich unless statedotherwise. MitoQ was kindly provided by M. P. Murphy (Cambridge, UK). Thecollection of screening compounds was purchased fromChemDiv, ChemBridge andEnamine. Necrostatin-1 was obtained from Enzo Life Sciences GmbH.

Cell lines. The TAM-inducible Gpx4−/− cells (Pfa1) were described previously11.Rip1-knockout cells were generously provided by J. Silke (Melbourne, Australia)32.Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% fetal bovine serum (FBS). Human renal proximal tubularepithelial cells (HRPTEpiCs; no. 4100) were purchased from ScienCell and culturedaccording to the manufacturer’s recommendations. HK-2 cells were cultured indefined keratinocyte-SFM-supplemented bovine pituitary extract and recombinantepidermal growth factor according to ATCC.

Cell viability assays and inhibitor studies. To induce the knockout of Gpx4,cells were seeded onto 96-well plates (1,000 cells per well) and treated with 1 µM4–OH-tamoxifen (TAM) after plating. Cell viability was assessed at different timepoints after treatment (usually 72 h, unless stated otherwise) usingAquaBluer, unlessstated otherwise, as an indicator of viable cells according to the manufacturer’srecommendations (MultiTarget Pharmaceuticals, LLC). Alternatively, cell death wasalso quantified by measuring released lactate dehydrogenase (LDH) activity usingthe Cytotoxicity Detection Kit (LDH) according to the manufacturer’s instructions(Roche Diagnostics Deutschland).

Immunoblotting. Western blot analysis of cell as well as tissue extracts wasperformed essentially as described previously11,33, by using antibodies against Gpx4(monoclonal rat antibody 1B4 (ref. 11) or 1:1,000, no. ab125066, Abcam), Rip3(1:1,000, no. hGTX107574, Genetex), Rip1 (1:1,000, no. 3493, Cell SignalingTechnologies) and ß-actin (1:1,000, no. A5441, Sigma).

Inducible disruption of Gpx4 in mice. The generation of mice with loxP-flanked(floxed, fl) Gpx4 alleles (Gpx4tm2Marc, in the following referred to as Gpx4fl/fl) waspreviously outlined in detail11. To bypass early embryonic lethality of Gpx4−/− mice,we took advantage of the TAM-inducible ROSA26–CreERT2 deleter strain (in thefollowing referred to as CreERT2) generously provided by A. Berns (The NetherlandsCancer Institute, Amsterdam,Holland)34. Thesemice carry a conditional Cre recom-binase (CreERT2) allele targeted to the ubiquitously expressed ROSA26 locus. Theoestrogen receptor T2 (ERT2) moiety fused to Cre retains the recombinase in thecytosol. Tamoxifen administration releases this inhibition, thus permitting induciblerecombination of loxP-flanked alleles. Gpx4fl/fl were first crossed with CreERT2

deleter mice to obtain CreERT2;Gpx4+/fl mice. CreERT2;Gpx4+/fl were then mated toGpx4fl/fl mice yielding CreERT2;Gpx4fl/fl mice. CreERT2;Gpx4wt/fl mice did not showany pathological alterations and therefore served as controls. To achieve inducibledisruption of the loxP-flanked Gpx4 allele(s), mice (>8 weeks of age; male and fe-male) were fed a diet containing tamoxifen citrate as described previously35 (n=19).

Generation of ALOX15/Gpx4 double-knockout mice. To generateAlox15/Gpx4 compound mutant mice, Alox15−/− (Alox15tm1Fun) mice (TheJackson Laboratory) were first crossed with CreERT2;Gpx4fl/fl mice to generateCreERT2;Gpx4+/fl /Alox15+/− mice. Subsequently, CreERT2;Gpx4+/fl /Alox15+/− weremated with Gpx4+/fl /Alox15+/− mice to obtain CreERT2;Gpx4+/fl /Alox15−/− andGpx4fl/fl /Alox15−/− mice. Finally, intercrossing of CreERT2;Gpx4+/fl /Alox15−/− micewith Gpx4fl/fl /Alox15−/− mice yielded CreERT2;Gpx4fl/fl /Alox15−/− mice. Disruptionof the floxed Gpx4 alleles was achieved by feeding mice (>8 weeks of age; male andfemale) with a diet containing tamoxifen citrate35 (n=8).

Isolation of CreERT2;Gpx4fl/fl/Alox15−/− and CreERT2;Gpx4fl/fl/Alox15+/+

cells. CreERT2;Gpx4fl/fl /Alox15−/− and CreERT2;Gpx4fl/fl /Alox15+/+ micewere euthanized by cervical translocation. After dissection, lungs wereminced and directly plated in standard DMEM containing 10% FBS andantibiotics as described previously11. CreERT2;Gpx4fl/fl /Alox15−/− (PZL cells)and CreERT2;Gpx4fl/fl /Alox15+/+ cells (PZ cells) were incubated at 5% oxygenand 5% CO2 in a water-saturated atmosphere for at least 20 passages to obtainspontaneously immortalized lung fibroblasts36.

Histological analysis.Kidneys were removed, trimmed free of adjacent tissues andweighed to the nearest 0.1mg. The kidneyswere cut into parallel slices perpendicularto the longitudinal axis, three 1mm3 cubes were taken from the kidney cortex andembedded in Epon for transmission electron microscopy, and the remaining tissuewas embedded in paraffin or plastic for light microscopy as described previously37.

Immunohistochemistry. Immunostaining was performed using 5-µm-thicksections of 4% paraformaldehyde-fixed paraffin-embedded tissue samples or

using 10-µm-thick sections of snap-frozen tissue samples. The following primaryantibodies were used on cryosections: rabbit anti-phospho-histone H3 antibody(1:200; no. 9701; Cell Signaling Technology, New England Biolabs GmbH) forproliferating cells, rat anti-F4/80 antibody (1:1,000; BM4007; Acris AntibodiesGmbH) for macrophages and rabbit anti-Cleaved Caspase-3 antibody (1:200; no.9664; Cell Signaling Technology, New England Biolabs GmbH) for detectionof apoptotic nuclei. Immunohistochemical staining for Gpx4 expression wasperformed on paraffin sections using a rabbit anti-Gpx4 antibody (1:250; no. 3649-1;Epitomics). As secondary antibodies a biotinylated rabbit anti-rat IgG (1:200;BA-4000; Vector Laboratories, Linaris, Wertheim-Bettingen) and a biotinylatedgoat anti-rabbit IgG (1:200; BA-1000; Vector Laboratories) were used. Staining wasperformed using the Vector ABC kit and Vector DAB kit (Vector Laboratories).

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick endlabelling (TUNEL). For the detection of TUNEL-positive cells, the ApopTagPeroxidase In SituApoptosis Detection Kit was used according to themanufacturer’sinstructions (S7100; Serologicals, Millipore).

Measurement of clinical parameters. Serum samples were analysed for chloride,creatinine and urea using the Architect ci8200 Autoanalyser (Abbott) and reagentssupplied by Abbott. The measurements were kindly performed at the City ClinicMunich in Schwabing, Munich, Germany.

Staining for lipid peroxidation in cells by flow cytometry. Cells were platedin 10 cm dishes in DMEM supplemented with 10% FBS and treated for definedtimes. After incubation, cells were washed and BODIPY 581/591 C11 (Invitrogen)or mito-BODIPY 581/591 C11 (kind gift from M. P. Murphy) was added for 1 h foranalysis by flow cytometry. Cells were trypsinized, washed and transferred to FACStubes in HBSS containing 2% FBS. Flow cytometry was performed using the BDFACS Canto II.

Synthesis of RSL3. Synthesis and characterization of RSL3 stereoisomers wereperformed according to ref. 8 with modifications for the installation of thechloroacetamide moiety as follows. The corresponding RSL3 intermediate (0.4 g,1.1mmol, 1.0 equivalent) was dissolved in 5ml of anhydrous CH2Cl2 andtriethylamine (0.17ml, 1.2mmol, 1.1 equivalent) was added. The reaction mixturewas cooled to 0 ◦C, after which a 0 ◦C solution of chloroacetyl chloride (0.1ml,1.2mmol, 1.1 equiv.) in 1.0ml CH2Cl2 was added dropwise. After 1 h at 0 ◦C, thereaction was quenched with aqueous saturated NH4Cl. The organic phase wasseparated, and the aqueous phase was extracted with 3×10ml CH2Cl2. The organicextracts were combined and washed with 30ml H2O and 30ml brine, dried withNa2SO4 and then concentrated to give the crude product as a yellow oil. Thecompound was purified by dry-loaded silica gel chromatography in a gradientelution from 100% hexane to 50:50 ethyl acetate/hexane. An identical protocol wasused to obtain the chloroacetamide derivative of the other three diastereomers. Thefour diastereomers were obtained in 80 to 90% yield.

Transmission electron microscopy of cells. Cell pellets were fixed in 2.5%electron microscopy grade glutaraldehyde in 0.1M sodium cacodylate buffer pH 7.4(Science Services), postfixed in 2% aqueous osmium tetraoxide, dehydrated ingradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck) andcured for 24 h at 60 ◦C. Semithin sections were cut and stained with toluidineblue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, andstained with uranyl acetate and lead citrate before examination by transmissionelectron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH). Pictures wereacquired using a SlowScanCCD(charge-coupled device) camera and iTEMsoftware(Olympus Soft Imaging Solutions).

siRNA and transfection protocol. Cells were plated at 25,000 cells per wellin a 6-well plate and transfected with Hyperfect according to the manufacturer’srecommendations (Qiagen). The cells were kept in the transfection mixture untilthe end of the assay. siRNAs for Ripk1, Ripk3 and Scp2 were purchased fromQiagen (no. GS19766, no. GS56532 and no. GS20280, respectively). Gpx4 siRNAwas obtained fromOn-Target Plus SMARTpool human GPx4 siRNA (no. L-011676-00-0005, Thermo Scientific Dharmacon).

Determination of intracellular GSH levels. Cells were seeded on a 6-well plate(1× 105 cells per well) and cultured for 24 h in the routine culture medium.After treatments with different inhibitors, cells were washed three times with ice-cold PBS, extracted with 5% trichloroacetic acid, and then treated with ether toremove the trichloroacetic acid. Total glutathione content in the aqueous layerwas measured using an enzymatic method, based on the catalytic action ofglutathione in the reduction of 5,5′-dithiobis (2-nitrobenzoic acid) by the glutathionereductase system38.

NATURE CELL BIOLOGY


DOI: 10.1038/ncb3064 METHODS

Measurement of hydroxyeicosatetraenoic acids by LC–MS/MS. To determinethe amount of HETEs released into the medium on Gpx4 deletion, cells were seededon 6-well dishes with or without treatment. Supernatants were collected at theindicated time points after treatment and analysed for the presence of arachidonicacid oxidation products.

For lipid extraction, PGE2-d4 and 15-HETE-d8 (5 ng)were added to each samplebefore extraction as an internal standard. Samples were also spiked with butylatedhydroxytolulene and DTPA (100 µM final concentration in samples). Lipids werethen extracted by adding a solvent mixture (1M acetic acid, 2-propanol, hexane(2:20:30)) to the sample at a ratio of 2.5ml solvent mixture to 1ml sample, vortexingand then adding 2.5ml of hexane. Following vortexing and centrifugation (1,000g ,5min), lipids were recovered in the upper hexane layer. The samples were then re-extracted by the addition of an equal volume of hexane followed by further vortexingand centrifugation. The combined hexane layers were then dried under vacuum andanalysed for 5-, 8-, 11-, 12- and 15-HETE using LC–MS/MS (as described below).

For free eicosanoid quantification using LC–MS/MS, samples were separatedon a C18 Spherisorb ODS2, 5 µm, 150 × 4.6-mm column (Waters) using agradient of 50–90% B over 20min (A, water/acetonitrile/acetic acid, 75:25:0.1; B,methanol/acetonitrile/acetic acid, 60:40:0.1) with a flow rate of 1mlmin−1. Productswere quantified by LC–MS/MS electrospray ionization on an Applied Biosystems4000 Q-Trap using parent-to-daughter transitions ofm/z 319.2 (HETE, [M-H]−) tom/z 115.1 (5-HETE), 155.1 (8-HETE), 167.1 (11-HETE), 179.1 (12-HETE), 219.1(15-HETE) andm/z 327.2 to 226.2 for 15-HETE-d8, with collision energies of−20to −30V. Products were identified and quantified using synthetic lipid standardsrun in parallel under the same conditions. Acquisition of product ion spectra wastriggered during elution of ions of interest, with the instrument operating in iontrap mode.

Oxidative lipidomics. Phospholipids/oxidized phospholipids were characterizedafter 2D-HPTLC separation39, followed by LC–MS analysis as described previously40.To assess phospholipid hydroperoxides and fatty acid hydroperoxides, lipid extractswere treated with a reductant, triphenylphosphine (10mgml−1, for 20min at4 ◦C in methanol)41, converting hydroperoxy- into respective hydroxy-species42. Allmass spectra were acquired in data-dependent negative ionization mode using aninclusion list for CL, PC and PE species, isolation width of 1.0 Da and high-energycollision of 24. The peaks with a signal-to-noise ratio of 3 and higher were takeninto consideration. Isotopic correction was performed by entering the chemicalcomposition of each species into the Qual browser of Xcalibur (operating system).PC and LPC molecular ions were detected as acetate adducts and formate adducts.Other phospholipids were detected as singly charged ions. Mass spectra of PC, PEand CLwere acquired using a QExactive orbitrapmass spectrometer (ThermoFisherScientific), whereby the resolution was set up at 140,000 with 5 ppm error. Themass spectra of LPC, LPE and mCL were acquired using an LXQ ion trap massspectrometer (ThermoFisher Scientific). Thus, them/z values are presented to threeand one decimal places, respectively. Standards for phospholipids and oxygenatedfatty acidswere fromAvanti Polar Lipids andCaymanChemicals. Standards ofmCLswere prepared as described previously43.

Phenotypic screening for ferroptosis inhibitors. In brief, compound seedingonto 96-well plates (1,000 cells per well) was carried out simultaneously with 1 µMTAM administration (leading to Gpx4 inactivation) obviating multiple mediumchanges, followed by incubation for 72 h. Cell viability was assessed subsequentlyusing the live/dead assay dye AquaBluer. In the primary screening round, allcompounds were tested at a single concentration of 10 µM and positive hits wereselected from wells with >80% cell viability. To confirm primary hits, compoundswere re-screened in the same assay and dose-dependent survival as well astoxicity curves were obtained using concentrations of 0–100 µM. IC50 and TC50

values were calculated using the GraphPad Prism software. Validated hits werethen evaluated based on efficacy, selectivity for ferroptosis, therapeutic range andphysicochemical properties. In addition, an in silico ADME-Tox screening wasimplemented to exclude compounds with potential in vivo side effects. To furthervalidate Liproxstatin-1, SAR studies were performed using commercially availablederivatives (ChemDiv).

Cytochrome P450 inhibition, hERG channel inhibition and pharmacokinet-ics studies. All experiments were performed by Cyprotex Discovery, according toestablished protocols.

HepG2 toxicity measurement. Cells were incubated with increasingconcentrations of compound for 72 h and cell viability was assessed aftertreatment using AquaBluer as an indicator of viable cells according to themanufacturer’s recommendations. TC50 values were calculated using the GraphPadPrism software.

Animal experiments (in vivo rescue experiment). Animals included in thetreatment study of inducible Gpx4−/− mice were equally distributed betweensex and weight, with typically 8–10 weeks of age. The average weight withinthe groups was between 22 and 24 g. Groups were formed to have comparablenumbers of females/males of the same age. Animal weight was arranged to have asimilar distribution between females and males. For the pharmacological inhibitorexperiments, CreERT2;Gpx4fl/fl mice were injected on day 1 and 3 with 0.5mg TAMdissolved in Miglyol. On day 4, compound treatment was started (Liproxstatin-1: 10mg kg−1) along with vehicle control (1% dimethylsulphoxide (DMSO) inPBS). Liproxstatin-1 and vehicle control were administered once daily by i.p.injection. Survival analysis was performed using the GraphPad Prism softwareand statistical analysis was done according to the log-rank (Mantel–Cox) test. Thecompounds, vehicle and Liproxstatin-1, were both odourless and colourless ensuringno detectable bias. Injections and daily animal assessment were performed in ablinded fashion. When animals showed terminal signs, they were euthanized. Nostatistical method was used to predetermine sample size for the treatment of theGpx4−/− mice. Mice were kept under standard conditions with food and water adlibitum (ssniff). All experiments were performed in compliance with the GermanAnimal Welfare Law and have been approved by the institutional committee onanimal experimentation and the government of Upper Bavaria.

Animal experiments (liver ischaemia/reperfusion). Animals (C57BL/6J) forthe liver ischaemia/reperfusion study were male mice between 8 and 10weeks of age, typically between 22 and 24 g and purchased from CharlesRiver. The animals were randomly distributed. Investigators performing theinjections/surgeries were blinded. All animals were euthanized 24 h followingtransient ischaemia/reperfusion. The surgical procedurewas performed as describedelsewhere44. In brief, C57BL/6J mice were narcotized with xylazine/ketamine andshaved at their front. After opening the abdominal cavity an atraumatic clip wasplaced across the portal vein, hepatic artery, and bile duct, just above branching tothe right lateral lobe. After 90min of ischaemia, the clamp was removed and theliver was reperfused. Blood and tissues were collected from anaesthetized animalsafter 24 h. Serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) were measured using a Dimension 1500 Vista Analyzer.

Data presentation and statistical analyses. Data are presented as mean ± s.d.unless stated otherwise. As a general rule for the cell-based experiments, the graphsshow the mean ± s.d. of n= x wells (x values are given in the figure legends)representative of a single experiment performed independently y times (y value isgiven in figure legends) for reproducibility. Statistical analysis was performed usingGraphPad Prism 5.0 software.

32. Wong, W. W. et al. RIPK1 is not essential for TNFR1-induced activation of NF-κB.Cell Death Differ. 17, 482–487 (2010).

33. Schneider, M. et al. Mitochondrial glutathione peroxidase 4 disruption causes maleinfertility. FASEB J. 23, 3233–3242 (2009).

34. Hameyer, D. et al. Toxicity of ligand-dependent Cre recombinases and generation ofa conditional Cre deleter mouse allowing mosaic recombination in peripheral tissues.Physiol. Genomics 31, 32–41 (2007).

35. Kiermayer, C., Conrad, M., Schneider, M., Schmidt, J. & Brielmeier, M. Optimizationof spatiotemporal gene inactivation in mouse heart by oral application of tamoxifencitrate. Genesis 45, 11–16 (2007).

36. Parrinello, S. et al. Oxygen sensitivity severely limits the replicative lifespan of murinefibroblasts. Nat. Cell Biol. 5, 741–747 (2003).

37. Herbach, N. et al. Diabetic kidney lesions of GIPRdn transgenic mice: podocytehypertrophy and thickening of the GBM precede glomerular hypertrophy andglomerulosclerosis. Am. J. Physiol. Renal. Physiol. 296, F819–F829 (2009).

38. Tietze, F. Enzymic method for quantitative determination of nanogram amounts oftotal and oxidized glutathione: applications to mammalian blood and other tissues.Anal. Biochem. 27, 502–522 (1969).

39. Rouser, G., Fkeischer, S. & Yamamoto, A. Two dimensional then layerchromatographic separation of polar lipids and determination of phospholipids byphosphorus analysis of spots. Lipids 5, 494–496 (1970).

40. Tyurina, Y. Y. et al. A mitochondrial pathway for biosynthesis of lipid mediators. Nat.Chem. 6, 542–552 (2014).

41. Tanaka, R., Hatate, H., Ito, M. & Nakamura, T. Elevation of lipid peroxide level andproduction of hydroxy lipids in cultured Hepa-T1 cells by oxidative stressors. FisheriesSci. 72, 665–672 (2006).

42. Tyurina, Y. Y., Tyurin, V. A., Epperly, M. W., Greenberger, J. S. & Kagan, V. E. Oxidativelipidomics of gamma-irradiation-induced intestinal injury. Free Radic. Biol. Med. 44,299–314 (2008).

43. Kim, J. & Hoppel, C. L. Monolysocardiolipin: improved preparation with high yield.J. Lipid Res. 52, 389–392 (2011).

44. Eggenhofer, E. et al. Unconventional RORgammat+ T cells drive hepatic ischemiareperfusion injury. J. Immunol. 191, 480–487 (2013).

NATURE CELL BIOLOGY


S U P P L E M E N TA RY I N F O R M AT I O N

WWW.NATURE.COM/NATURECELLBIOLOGY 1

DOI: 10.1038/ncb3064

b a

c d

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Angeli_Supplementary Fig. 1

CreERT2;Gpx4fl/fl CreERT2;Gpx4+/fl

a-C

asp-

3 F4

/80

CreERT2;Gpx4fl/fl CreERT2;Gpx4+/fl

CreERT2;Gpx4fl/fl/Alox15-/-

H &

E

PAS

CreERT2;Gpx4fl/fl/Alox15+/+ CreERT2;Gpx4+/fl/Alox15-/-

TAM [h] 0 24 48 72 96 testis

Gpx4

ß-actin

19 kDa

45 kDa

g

Supplementary Figure 1 Genetic loss of Alox15 does not rescue cell death and ARF induced by Gpx4 inactivation. (a) TAM-inducible Gpx4 deletion in lung fibroblasts derived from CreERT2;Gpx4fl/fl/Alox15-/- mice (PZL cells). (b) PZ (Alox15-proficient) and PZL (Alox15-deficient) lung fibroblasts can be rescued from Gpx4 deletion-induced cell death by lipophilic (αToc), but not by hydrophilic antioxidants (NAC, N-acetylcysteine). Gpx4 knockout was induced by the addition of 1 µM TAM in the presence of increasing concentrations of the antioxidants; cell viability was measured 72h later. (c,d) Knockdown of Alox5 delays cell death upon Gpx4 deletion only in cells additionally lacking Alox15 (PZL) (d), but not in cells proficient for Alox15 (PZ) (c). Cell death was monitored at different time points after KO induction using AquaBluerTM. Knockdown efficiency was monitored by quantitative PCR. Data shown represents the mean ± S.D. of n= 3

wells (b,c,d) of a 96-well plate (or 6-well plate for RNA isolation) from a representative experiment performed independently three times, ANOVA (*p < 0.05). (e) Histological analysis of kidneys of TAM-treated CreERT2;Gpx4fl/

fl/Alox15-/- mice revealed widespread tubular cell death, interstitial edema, and proteinaceous casts in distal tubules like TAM-treated CreERT2;Gpx4fl/

fl/Alox15+/+ mice (scale bars 100 µm) (Periodic Acid-Schiff Stain, PAS). (f) Electron micrographs confirmed the severe destruction of renal tubule cells in symptomatic CreERT2;Gpx4fl/fl/Alox15+/+ mice (scale bars 10 µm). (g) Staining against F4/80, a 160 kD glycoprotein expressed by murine macrophages, revealed interstitial inflammatory cell infiltrates in response to massive tissue injury in symptomatic Gpx4 null kidneys (CreERT2;Gpx4fl/fl). Staining against active caspase-3 (a-Casp-3) showed faint staining in kidney tissue of Gpx4 knockout mice.




2 WWW.NATURE.COM/NATURECELLBIOLOGY

a b

c d

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Supplementary Figure 2 Caspases are not involved in ferroptotic cell death. (a) The pan caspase inhibitor Z-VAD-FMK (zvad) does not prevent cell death triggered by Gpx4 deletion. Knockout of Gpx4 was induced by adding 1 µM TAM in the presence of increasing zvad concentrations and cell viability was determined 72h later. Data shown represents the mean ± S.D. of n= 3 wells of a 96-well plate from a representative experiment performed independently three times. (b) Inducible Gpx4 deletion in Pfa-1 cells does not lead to caspase activation. Caspase activity was determined using CellEvent™ Caspase-3/7 Green Detection Reagent (Life Technologies, Thermo Fisher Scientific) at different time points after KO induction. (c) Ferroptosis inducers do not increase caspase activity. Activities were determined in Pfa-1 cells after treatment with the different inducers at specific time points [TAM (1 µM – 48h), BSO (10 µM – 48h), erastin (1 µM – 12h), RSL3 (0.5 µM – 3h), staurosporine (Stauro; 0.2 µM – 6h) and TNFα (10 ng/ml – 9h)]. (d) Class I FINs (BSO and erastin) decrease GSH levels, whereas the class II FINs RSL3 and Gpx4 do not impact on GSH levels. Total GSH levels were determined after treatment with the different inducers at specific time points [BSO (10 µM – 24h), erastin (1 µM – 8h), RSL3 (0.5 µM – 3h) TAM (1 µM – 48h).

Data shown (b,c,d) represents the mean ± S.D. of n= 3 wells of a 6-well plate from a representative experiment performed independently two (b,c) or three (d) times. (e) Kinetics of cell death induced by ferroptosis-inducing agents and apoptosis-inducing agents in Pfa1 cells. Cells were treated with BSO (20 µM), erastin (2 µM), RSL3 (0.5 µM), TAM (1 µM), Staurosporine (0.5 µM) and TNFα (10 ng/ml). Cellular viability was assayed using AquaBluerTM. (f) Profile of rescue of Gpx4 proficient Pfa1 cells treated with apoptotic and ferroptotic cell death triggers. Cells were treated with the cell death inducers for the indicated times [TAM (1 µM – 72h), erastin (1 µM – 24h), RSL3 (0.5 µM – 24h), TNFα (10 ng/ml – 48h) and Staurosporine (0.2 µM – 48h)]. The rescue profile was performed using fixed concentrations of Z-VAD-FMK (zvad, 50 µM), Nec1 (25 µM), αToc (2 µM), Fer1 (0.2 µM) and DFO (10 µM), erastin (1 µM – 12h), RSL3 (0.5 µM – 3h), Staurosporine (0.2 µM – 6h) and TNFα (10 ng/ml – 9h). (g,h) Overexpression of active Gpx4 (WT), but not Gpx4-U46S in Gpx4-proficient Pfa1 cells, increases resistance to RSL3- and erastin-induced ferroptosis. Data shown represents the mean ± S.D. of n= 3 (e,f) or n=4 wells (g,h) of a 96-well plate from a representative experiment performed independently three times.




b

c

c

d

ß-actin

Rip3

DMSO TNFα Stauro BSO Erastin TAM (Gpx4 KO)

55 kDa

45 kDa

a


Supplementary Figure 3 Receptor protein interacting kinases 1 and 3 do not contribute to ferroptotic cell death. (a) An immunoblot against Rip3 showing that Rip3 is only weakly expressed in Pfa1 cells in contrast to L929 fibroblasts. (b) Rip1 knockout (KO) cells do not show increased resistance to the ferroptosis inducing agents as compared to Rip1 wildtype (WT) cells. Cell death was assessed 24h or 48h after treatment with erastin or BSO, respectively (top row). Nec1 prevents cell death induced by ferroptosis inducers even in Rip1 KO cells. Viability was assessed after adding increasing concentrations of Nec1 (0-50 µM) to Rip1 KO cells

treated with 10 µM BSO or 0.4 µM erastin (bottom row). (c) Ultrastructural analysis of Gpx4-proficient Pfa-1 cells treated with various FINs, classical inducers of apoptotic cell death and upon Gpx4 deletion (TAM) (Scale bars are 2 µm in the top row and 200 nm in the bottom row). (d) MitoQ and DecylQ (1 µM) prevented cell death induced by Gpx4 deletion, an effect that is not modulated by dissipating the mitochondrial membrane potential with trifluorocarbonylcyanide phenylhydrazone (FCCP, 1 µM). Data shown represents the mean ± S.D. of n= 4 wells (d) or n= 6 wells (b) of a 96-well plate from an experiment performed independently three times.




a b

c

d e


Supplementary Figure 4 Knockdown of Alox5 slows down the release of HETEs and delays cell death kinetics in Alox15/Gpx4 double knockout cells. (a,b) Quantification of released arachidonic acid metabolites (5-HETE, 11-HETE, 12-HETE and 15-HETE) was performed by LC/MS/MS from supernatants collected at 24, 48 and 72h after Gpx4 KO induction (PZ cells are wildtype for Alox15, whereas PZL cells are knockout for Alox15). Data shown represents the mean ± S.D. from one well of a 6-well plate, pooled from n= 3 repeated two times. (c) PZL cells stably transduced with an shRNA against Alox5 are less susceptible to ferroptotic stimuli than the parental PZL and PZ cells, whereas all the cell lines showed similar sensitivity to staurosporine-induced apoptosis and tBOOH-induced necrosis.

(Data shown represents the mean ± S.D. from one well of a 96-well plate, pooled from n= 3 (c) representative experiment performed three times, ANOVA (*p < 0.05). (d) siRNA-mediated knockdown of SCP2 partially protects against cell death in response to Gpx4 deletion. Shown are experiments performed with three independent siRNAs (Scp2_1, Scp2_2, Scp2_3) (Scb, scrambled siRNA). (e) Kinetics of cell death upon Gpx4 deletion and SCP-2 knockdown (Scp2_2). Pfa1 cells were induced and transfection with the siRNA was performed 6h thereafter, and viability was determined at different time points. Data shown represents the mean ± S.D. of n= 3 wells (e) or n= 4 wells (e) of a 6- (e) or 12- (e) well plate from an experiment performed independently three times, ANOVA (*p < 0.05).




100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z

255.232

774.5994 283.265

311.235 536.3720

93.6946 168.0419 461.8188 848.570 [M+CH3COOH – H]-

[M-CH3 – H]-

[18:2-OOH – H]-

[18:0 – H]-

[16:0 – H]-

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z

279.232

255.232

742.537 480.3091

168.0422 462.292 504.3089

816.537

[M – CH3 – 18:2]-

[M – CH3 – 18:2+H2O]-

[M – CH3 – 16:0+H2O]-

[M-CH3 – H]-

[M+CH3COOH – H]-

[18:2 – H]-

[16:0 – H]-

150 200 250 300 350 400 450 500 550 600 650 700 750 m/z

303.232

283.264 766.539 480.309

462.300 196.037

140.010

[20:4 – H]-

[M – 20:4]-

[18:0 – H]-

[M – H]- [M – 20:4+H2O]-

335.222

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 m/z

283.264 61.9867

798.538 480.309

140.010 196.036 462.298

[20:4-OOH – H]-

[M – H]-

[18:0 – H]-

[M – 20:4-OOH+H2O]-

[M – 20:4-OOH]-

150 300 450 600 750 900 1050 1200 1350 m/z

279.232

415.224

695.464

152.994 433.235 1447.961 831.457

751.491

200 400 600 800 1000 1200 1400 m/z

279.232

727.452 415.225

695.464 152.994

447.213 1479.932 1217.461

311.223

796.174 564.319

295.226 m/z 279.232 + 15.999 = m/z 295.226 m/z 279.232 + 31.998= m/z 311.223

[18:2 – H]-

[M – H]-

[M – H]-

[18:2-OH – H]-

[18:2-OOH – H]-

[18:2 – H]-

1455 1470 1485 1500 1515 1530 1545 m/z

1499.947 1463.976 1497.938

1465.989

1479.932

1525.9651

1481.975

1455 1470 1485 1500 1515 1530 1545 m/z

1481.972

1463.969 1507.988 1465.986 1529.971 1499.951

1479.940

CL-‐OOH

CL-‐OH

(18:2)3(18:2-OOH)1 (18:1)1(18:2)2(18:2-OOH)1

+TPP

c

a

b

d

0

5

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0 0.5

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2 2.5

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-OO

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PC

(16:0)(22:6-OOH) (18:1)(20:4-OOH) (18:0)(18:2-OOH) (18:2)(18:2-OOH)

- wt/fl - fl/fl

* * *

*


Supplementary Figure 5 Oxi-lipidomics of Gpx4-/- kidneys. (a) MS2 fragmentation patterns of non-oxidized PC with m/z 816.537 (upper panel) and its oxygenated species with m/z 848.570 (lower panel). PCs were detected as acetate adducts. The levels of hydroperoxy PC species in kidney from Gpx4 null and wildtype mice. Hydroperoxy PC species (18:2/18:2-OOH, m/z 872.575), (18:0/18:2-OOH, m/z 876.606), (16:0/22:6-OOH, m/z 896.575) and (18:1/20:4-OOH, m/z 898.587) were originated from PC species (18:2/18:2, 840.576), (18:0/18:2, m/z 844.607), (16:0/22:6, m/z 864.576) and (18:1/20:4 m/z 866.588). PC hydroperoxy-species are

presented as pmol/nmol of the parental non-oxidized PC species. Data is mean ± S.D. of n= 7 pooled animals from two independent experiments, *p >0.05 vs. wildtype. (b) MS2 fragmentation patterns of non-oxidized PE with m/z 766.539 (upper panel) and its oxygenated species with m/z 798.538 (lower panel). (c) MS2 fragmentation patterns of non-oxidized CL (upper panel) and its oxygenated species (lower panel). (d) Mass spectra of oxygenated CL before (left upper panel) and after reduction with TPP fragmentation patterns of non-oxidized CL (left upper panel) and representative structures of hydroperoxy- (right upper panel) and hydroxy- (right lower panel) species.




b


Supplementary Figure 6 Specificity of Liproxstatin-1 against ferroptotic cell death. For all the inhibitor assays, the cells were pre-incubated with Liproxstatin-1 (500 nM) for 24h. Then, each lethal compound was added at increasing concentrations for another 48h, and cell viability was

assessed using AquaBluerTM. IC50 depicted in the figures represent the lethal compound LD50 in the presence or absence of Liproxstatin-1. Data shown represents the mean ± S.D. of n= 4 wells of a 96-well plate from an experiment performed one time (nd=not determined).




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Gpx

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1- Pfa1 2- O.E Gpx4 3- HK2 4- HRPEitC 5- Kidney

1 2 4 5 3

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t lo

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19kDa 24kDa

19kDa 24kDa

ß-actin

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Gpx4

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Supplementary Figure 7 Gpx4 inactivation induces ferroptosis in human kidney cells. (a) Specificity of active (1S,3R)-RSL3 and inactive (1R,3R)-RSL3 on cell death induction in Pfa1 cells and Pfa1 cells overexpressing Gpx4. Cells were treated with increasing concentrations of both isomers (0-0.5 µM), and cell viability was assessed 24h thereafter. (b) Impact of active and inactive forms of RSL3 on the viability of primary (HRPEitC) and transformed (HK-2) human tubular epithelial kidney cells. Cells were treated with increasing concentrations of the two isomers (0-0.5 µM), and cell viability was assessed 48h thereafter. (c) Gpx4 knockdown induces cell death of human kidney tubular cells (HK-2). Impaired viability due to Gpx4 knockdown is rescued by αToc (10 µM) in HK-2 cells (scb, scrambled). (d) Kidney

tubular cells (lanes 3, 4) show remarkably high expression levels of Gpx4 that are even higher than in Gpx4 overexpressing Pfa1 cells (lane 1). Gpx4 in overexpressing cells (lane 2) is visualized at approximately 24 kDa due to the presence of an N-terminal tandem affinity purification enhanced (TAPe) tag. (e) siRNA-mediated downregulation of Gpx4 sensitizes to (1S,3R)-RSL3 but not (1R,3R)-RSL3 treatment. Cells were transfected with 20 nmol of siRNA targeting Gpx4 and treated with the compounds 24h after transfection. Cell viability was determined 24h after treatment with the chemicals. Data shown in the individual sub-panels represents the mean ± S.D of n= 3 wells (a, b and e) or n= 4 wells (c) of a 96- (a,b and e) or 6-well (c) plate from a representative experiment performed independently at least two times, ANOVA (*p < 0.05).




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Gpx4

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In house Gpx4 an.body


ß-actin

Gpx4

Fig 1E

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19kDa 24kDa

Gpx4

Gpx4

Abcam (ab125066) Gpx4 an.body

Sup Fig

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Fig S7D

Gpx4

β-actin

siRNA


Fig S7C

Supplementary Figure 8 Scans of uncropped blots.




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+/fl,Cre fl/fl,Cre

Gpx4

ß-tubulin



ß-actin

Gpx4

Fig 1E

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45

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25 35

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55

Fig S1A

1 2 4 5 3

Sho

rt lo

ng

ß-actin

19kDa 24kDa

19 24

19kDa 24kDa

Gpx4

Gpx4


Sup Fig

45

Fig S7D

Gpx4

β-actin

siRNA


Fig S7C

Creatinine Urea Chloride(mg/dl) (mg/dl) (mmol/l)

Gpx4 (+/fl, Cre) 0.42 + 0.03 54 + 6 114 + 5Gpx4 (fl/fl, Cre) 0.59 + 0.22* 108 + 61* 111 + 10

Supplementary Table 1 Clinical chemical parameters of serum. Serum levels of creatinine, urea and chloride are shown in inducible Gpx4 knockout and control animals.


Inactivation of the ferroptosis regulator Gpx4 …...preventing lipid-oxidation-induced acute renal...

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