Chaperone-mediated autophagy is involved in theexecution of ferroptosisZheming Wua,b, Yang Genga, Xiaojuan Lua, Yuying Shia, Guowei Wua,b, Mengmeng Zhanga, Bing Shana, Heling Pana,1,and Junying Yuanc,1

aInterdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 201203 Shanghai,China; bUniversity of Chinese Academy of Sciences, 100049 Beijing, China; and cDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115

Contributed by Junying Yuan, December 14, 2018 (sent for review November 26, 2018; reviewed by Andreas Linkermann and Adrian T. Ting)

Necroptosis and ferroptosis are two distinct necrotic cell deathmodalities with no known common molecular mechanisms. Nec-roptosis is activated by ligands of death receptors such as tumornecrosis factor-α (TNF-α) under caspase-deficient conditions, whereasferroptosis is mediated by the accumulation of lipid peroxides uponthe depletion/or inhibition of glutathione peroxidase 4 (GPX4). Themolecular mechanism that mediates the execution of ferroptosisremains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heatshock protein 90 (HSP90), as an inhibitor of necroptosis that couldalso inhibit ferroptosis. We found that HSP90 defined a commonregulatory nodal between necroptosis and ferroptosis. We showedthat inhibition of HSP90 by CDDO blocked necroptosis by inhibitingthe activation of RIPK1 kinase. Furthermore, we showed that theactivation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediatedautophagy (CMA), which, in turn, promoted the degradation ofGPX4. Importantly, inhibition of CMA stabilized GPX4 and reducedferroptosis. Our results suggest that activation of CMA is involvedin the execution of ferroptosis.

necroptosis | ferroptosis | HSP90 | CMA | RIPK1

Programmed cell death (PCD) is involved in mediating diverseaspects of development, homeostasis, and diseases in multi-

cellular organism (1–3). Caspase-dependent apoptosis was thefirst well-characterized form of PCD (4). While necrosis hadtraditionally been considered to be an uncontrolled processtriggered by overwhelming environmental stress or acute injury,necroptosis (5) and ferroptosis (6) have now been shown to betwo distinct regulated necrosis pathways that can be activated indifferent conditions and may function under diverse physiologi-cal and pathological contexts.Necroptosis is mediated by the kinase activity of receptor

interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineagekinase domain-like pseudokinase (MLKL) (7). Tumor necrosisfactor-α (TNF-α) remains the best understood and most im-portant trigger of necroptosis under various pathological condi-tions in humans. In response to the activation of TNF receptor(TNFR) family members, RIPK1 is recruited to the cytosolic sideof the receptor and activated (8). Activated RIPK1 kinase, inturn, interacts with RIPK3 kinase (9–11) to mediate the re-cruitment and phosphorylation of MLKL (12). PhosphorylatedMLKL forms oligomers and translocates to the plasma mem-brane, where it disrupts membrane integrity to mediate necroticcell death (13). In TNF-α–stimulated cells, RIPK1 functions as acritical cellular signaling hub to coordinate multiple dynamic reg-ulatory events in an RIPK1 kinase-dependent and -independentmanner, including nuclear factor κB (NF-κB) activation, apoptosis,and necroptosis (14–16). Inhibition of RIPK1 kinase blocks nec-roptosis and RIPK1-dependent apoptosis but has no effect on NF-κB activation.Distinct from necroptosis, ferroptosis is a form of iron-dependent

necrosis characterized by the formation of lethal lipid peroxidation(17, 18). Ferroptosis is remarkably distinct from necroptosis and

other forms of regulated cell death at biochemical and morphologicallevels. Ferroptosis was first identified in the research of cell deathinduced by the small molecule erastin or glutamate, which inhibitsactivity of cystine-glutamate antiporter (system Xc−) (6, 19). Ferroptosiscan be activated by treatment with erastin or high extracellularglutamate to lead to depletion of cysteine, inhibition of glutathione(GSH) synthesis, and inactivation of the phospholipid peroxidaseglutathione peroxidase 4 (GPX4), an enzyme crucial in convertingtoxic lipid hydroperoxides to nontoxic lipid alcohols (20). In-activation of GPX4 by direct inhibition with (1S, 3R)-RSL3 ordepletion of GPX4 with erastin results in overwhelming lipidperoxidation and cell death. However, the molecular mechanismthat mediates the execution of ferroptosis is unclear.Heat shock protein 90 (HSP90), an evolutionary conserved

and ubiquitously expressed molecular chaperone, plays an im-portant role in maintaining the function and stability of cellularproteins (21, 22). HSP90 has diverse functions in assisting fold-ing, intracellular transport, maintenance, and degradation ofproteins, as well as in facilitating cell signaling (23, 24). The clientproteins of HSP90 include kinases, transcription factors, and anumber of signaling molecules. Importantly, recent studies reveal thatRIPK1, RIPK3, and MLKL, the key regulators in the necroptosispathway, are client proteins of HSP90 (25–30). However, the role ofHSP90 on RIPK1 kinase activity is unclear, and we do not know theinvolvement of HSP90 in other form of cell death such as ferroptosis.Chaperone-mediated autophagy (CMA) is a cellular lysosome-

mediated degradative mechanism (31). CMA delivers selectedprotein substrates with a pentapeptide CMA-targeting motif intothe lysosome mediated by their bindings with the chaperone

Significance

This study reveals a common regulatory mechanism mediatedby HSP90 that is shared by necroptosis and ferroptosis, twonecrotic cell death mechanisms that have previously had noknown mechanistic connections. Furthermore, this study dem-onstrates the involvement of chaperone-mediated autophagyin the execution of ferroptosis. Given the role of HSP90 asan important chaperone involved in major human maladiesranging from neurodegenerations to cancers, our work furtherimplicates the roles of necroptosis and ferroptosis in humandiseases.

Author contributions: Z.W., B.S., H.P., and J.Y. designed research; Z.W., X.L., Y.S., G.W.,M.Z., B.S., and H.P. performed research; Y.G. contributed new reagents/analytic tools;Z.W., Y.G., B.S., H.P., and J.Y. analyzed data; and Z.W., H.P., and J.Y. wrote the paper.

Reviewers: A.L., Technical University Dresden; and A.T.T., Icahn School of Medicine atMount Sinai.

Conflict of interest statement: J.Y. is a consultant of Denali Therapeutics, Inc. J.Y. and A.L.are coauthors on two review articles published in 2016 and 2018.

Published under the PNAS license.1To whom correspondence may be addressed. Email: panheling@sioc.ac.cn orjunying_yuan@hms.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1819728116/-/DCSupplemental.

Published online February 4, 2019.

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HSC70 and the interaction with lysosome-associated membraneprotein type 2a (Lamp-2a). CMA is implicated in cellular proteinquality control by mediating the degradation of damaged proteins(32). The role of CMA in any specific form of cell death is unclear.Here, we identify a triterpenoid compound, 2-amino-5-chloro-

N,3-dimethylbenzamide (CDDO), which is known to inhibitHSP90, as an inhibitor of both necroptosis and ferroptosis. Weshow that CDDO and inhibition of HSP90 block the kinase ac-tivity of RIPK1 and the formation of complex IIb, a criticalcomplex involved in the execution of necroptosis. Furthermore,treatment with CDDO also inhibits GPX4 degradation by block-ing CMA through HSP90 inhibition to block ferroptosis. Thus,our results demonstrate that HSP90 serves as a common regula-tory nodal of both necroptosis and ferroptosis, and suggest thatCMA is involved in mediating execution of ferroptosis.

ResultsTriterpenoid CDDO Inhibits both Necroptosis and Ferroptosis. Toidentify new regulators of necroptosis, we performed a chemicalscreen with a library of ∼600 known bioactive compounds. Thescreen was carried out using a hippocampal-derived HT-22 cellline treated with a combination of TNF-α, zVAD.fmk, and thesmall molecule inhibitor of apoptotic proteins antagonist SM-164 (5). In this screen, we identified the triterpenoid com-pound CDDO (also known as bardoxolone) (SI Appendix, Fig.S1A) as a potent inhibitor of necroptosis in a time-dependentmanner (Fig. 1A). CDDO inhibited necroptosis of HT-22 cellsdose-dependently, with an EC50 of 10 μM (Fig. 1B). We testedthe ability of CDDO to block necroptosis in additional cellmodels and found that CDDO effectively inhibited necroptosisof mouse neural cell line 661W cells and human colon cancer cellline HT-29 cells induced by TNF-α/zVAD/SM-164, as well asnecroptosis of human leukemia FADD-deficient Jurkat cells in-duced by TNF-α only, TNF-α/SM-164, or TNF-α/cycloheximide(CHX) (SI Appendix, Fig. S1 B–D).

We next investigated the effect of CDDO on other forms ofcell death, including ferroptosis induced by glutamate and era-stin, TNF-α–induced apoptosis, DNA damage-induced apopto-sis, and oxidative stress-induced necrosis. Interestingly, we foundthat CDDO could also inhibit ferroptosis of HT-22, 661W, andhuman HT-1080 cells induced by glutamate and erastin, withEC50s of 5.5 μM and 6.8 μM, respectively (Fig. 1 C–E and SIAppendix, Fig. S1 E and F). The inhibitory effect of CDDO onboth ferroptosis and necroptosis was further confirmed using apropidium iodide (PI) uptake assay (Fig. 1F). However, the ad-dition of CDDO sensitized HT-22 cells to apoptosis induced byTNF-α, TNF-α/SM-164, and staurosporine, while it had no effect onapoptosis induced by various agents that included TNF-α–mediatedRIPK1-independent apoptosis induced by TNF-α/CHX, DNAdamage induced apoptosis by doxorubicin or etoposide, ER stress-induced apoptosis by thapsigargin or tunicamycin, and necrosis in-duced by oxidative stress using tert-butyl hydroperoxide (t-BOOH)or H2O2 (SI Appendix, Fig. S1G). Thus, CDDO can inhibit bothnecroptosis and ferroptosis, two necrotic cell death pathways thathave no known common regulatory mechanism, but not apoptosis.Since ferroptosis is sensitive to cellular redox status (6), we

next explored if the effect of CDDO on cell death was due to itsintrinsic antioxidant potential. We performed a 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay to monitor the antioxidant activityof CDDO. We found that unlike other inhibitors of ferroptosissuch as ferrostatin-1 (Fer-1), which is known to display a strongantioxidant effect with high free radical scavenging activity (33),CDDO did not demonstrate any antioxidant activity (SI Appen-dix, Fig. S1H). Thus, CDDO is a chemical inhibitor of ferroptosisand necroptosis but not an antioxidant, and it may be useful as aprobe to investigate the mechanism that is in common betweennecroptosis and ferroptosis.

CDDO Inhibits the Activation of RIPK1 Kinase Activity in Necroptosis.Next, we characterized the effects of CDDO on the biochemicalhallmarks of necroptosis and ferroptosis. We first analyzed the

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Fig. 1. Triterpenoid CDDO inhibits both necroptosis and ferroptosis. (A) HT-22 cells were pretreated with or without 10 μM CDDO or 10 μM Nec-1s for 30 minand then treated with 20 ng/mL TNF-α (T), 20 nM SM-164 (S), and 20 μM zVAD.fmk (Z) for 3, 6, or 9 h. Cell viability was measured by CellTiterGlo assay.(B) Dose–response curve (in micromole) of CDDO in HT-22 cells treated with T/S/Z for 9 h. (C) HT-22 cells were pretreated with CDDO and then treated with20 ng/mL TNF-α plus 20 μM zVAD.fmk with or without 20 nM SM-164 or 1 μg/mL CHX (C) for 9 h or 50 mM glutamate or 10 μM erastin for 12 h. Cell viabilitywas measured by CellTiterGlo assay. Ctrl, control. (D and E) Dose–response curve (in micromole) of CDDO cytoprotection in HT-22 cells treated with 10 μMerastin or 50 mM glutamate for 12 h. (F) HT-22 cells were pretreated with 10 μM CDDO and then treated with erastin (10 μM) for 8 h or T/S/Z for 4 h. Cell deathwas measured by PI uptake assay. Results shown are averages of triplicates ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001.

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effect of CDDO on specific phosphorylation events on RIPK1,RIPK3, and MLKL, including p-S166–RIPK1, p-T231/S232–RIPK3, and p-S345–mMLKL/p-S358–hMLKL, the biomarkersof their activation, during necroptosis (34–36). We found thatthe addition of CDDO strongly inhibited the induction of p-S166–RIPK1, p-RIPK3, and p-MLKL of HT-22 cells and HT-29 cells during necroptosis induced by TNF-α/SM-164/zVAD.fmk (Fig. 2A and SI Appendix, Fig. S2A).Since RIPK1 is an upstream activator of RIPK3 and MLKL, we

next tested if the formation of complex IIb, defined by the inter-acting complex of RIPK1 and RIPK3, was disrupted by CDDO.As shown in Fig. 2B, CDDO strongly inhibited the interaction ofRIPK1 and RIPK3 in HT-22 cells stimulated by TNF-α/SM-164/zVAD.fmk. These results suggest that CDDO inhibits the acti-vation of RIPK1 kinase, which, in turn, blocks the formation ofcomplex IIb, as well as the phosphorylation of RIPK3 and MLKL.To explore the mechanism by which CDDO inhibits RIPK1

kinase activity, we next investigated the effect of CDDO inTNFR1 signaling complex (TNF-RSC, complex I) in TNF-α–stimulated cells. RIPK1 was rapidly recruited into TNF-RSCwithin 5 min of TNF-α stimulation, which was not affected bythe cotreatment with CDDO (SI Appendix, Fig. S2B). Consistentwith the report that CDDO could inhibit the activity of inhibitorof NF-κB kinase subunit β (IKKβ) (37), we found that thetreatment with CDDO slightly reduced the phosphorylation ofIKK-α/β in complex I (SI Appendix, Fig. S2B) and the activationof NF-κB, as the phosphorylation and degradation of inhibitor ofNF-κB (IκBα) were also only slightly inhibited (SI Appendix, Fig.S2C). These results suggest that CDDO does not have a strongeffect on the activation of NF-κB.Since CDDO was highly effective in inhibiting the activation of

RIPK1, we next tested if CDDOmay inhibit the activation of RIPK1

directly. Interestingly, we found that CDDO markedly inhibited thephosphorylation of S166 RIPK1, a biomarker for activated RIPK1(8, 38), on overexpressed RIPK1 in 293T cells (Fig. 2C). More-over, we found that CDDO inhibited RIPK1 kinase activity in HT-22 cells induced by different necroptosis inducers (Fig. 2D).However, CDDO could not inhibit RIPK1 activity in an in vitrokinase assay, different from R-7-Cl-O-Nec-1 (Nec-1s) (SI Appen-dix, Fig. S2D). Based on these results, we concluded that CDDOwas an indirect inhibitor of RIPK1 kinase activity.

CDDO Inhibits Ferroptosis by Blocking GPX4 Degradation. To explorethe mechanism by which CDDO inhibits ferroptosis, we analyzedthe effect of CDDO on the biomarkers of ferroptosis, includingreactive oxygen species (ROS) accumulation, lipid peroxidation,and GPX4 degradation. We first characterized the effect ofCDDO on ROS levels in the HT-22 cell lines using 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), a cytosolic ROS sensor. Interestingly, the inductionof ROS in ferroptosis by erastin was significantly inhibited in thepresence of CDDO (Fig. 3A).Lipid peroxidation has been identified to be directly involved

in mediating necrosis and ferroptosis (17). We next investigatedwhether CDDO affected the production of malondialdehyde(MDA; an end product of lipid peroxidation) in ferroptosis in-duced by glutamate and erastin. We found that CDDO washighly effective in inhibiting the production of MDA, similar tothat of positive control Fer-1 (Fig. 3B). These results suggestedthat CDDO acted upstream of lipid peroxidation.Degradation of GPX4, a critical inhibitor of lipid peroxidation,

is important for promoting lipid peroxidation in ferroptosis (39).We next investigated the effect of CDDO on GPX4 degradationduring ferroptosis. We found that the treatment of HT-22 cellswith ferroptosis inducers such as erastin and glutamate led to areduction in the levels of GPX4 protein, which was blocked in thepresence of CDDO (Fig. 3C and SI Appendix, Fig. S3A). In ad-dition, inhibition of GSH production by L-buthionine-sulfoximine(BSO) can, in turn, impair GPX4 activity and promote ferroptosis(40). The addition of CDDO was also able to protect ferroptosisand inhibit the degradation of GPX4 induced by BSO (Fig. 3 Dand E). On the other hand, ferroptosis can also be induced di-rectly by knocking down GPX4 (39). However, unlike Fer-1,CDDO could not inhibit ferroptosis induced by direct knock-down of GPX4 (Fig. 3F and SI Appendix, Fig. S3B). Since theactivity of GPX4 is crucial for preventing ferroptosis, CDDOcannot inhibit type 2 ferroptosis induced by RSL3, a covalentinhibitor of GPX4, even though the protein level of GPX4 was alsorestored in the presence of CDDO (SI Appendix, Fig. S3 C and D).Thus, restoring the levels of GPX4 when its activity is inhibited by acovalent inhibitor, RSL3, is not sufficient to block ferroptosis. Thisresult suggests that CDDO protects erastin/glutamate-inducedferroptosis by preserving physiological levels of active GPX4.To determine if CDDO affects the transcription level of

GPX4, we measured the levels of GPX4 mRNA and confirmedthat induction of ferroptosis by glutamate/erastin had no effecton the levels of GPX4 mRNA. Moreover, the addition of CDDOalso had no influence on GPX4 mRNA levels in HT-22 cells withor without glutamate/erastin (SI Appendix, Fig. S3E).Taken together, we conclude that CDDO is a potent inhibitor

of ferroptosis that can block GPX4 degradation, lipid perox-idation, and ROS accumulation.

CDDO Targets HSP90 to Inhibit both Necroptosis and Ferroptosis.CDDO has been shown to inhibit multiple proteins, includingPPAR-γ, ErbB2, Akt, STATs, Keap1, and HSP90 (41). We nextevaluated the relevance of these targets in necroptosis and fer-roptosis using their inhibitors directly. Among all of the inhibi-tors we tested, we found that only Keap1 inhibitor tertiarybutylhydroquinone (t-BHQ), which can activate the nuclear

Fig. 2. CDDO inhibits the kinase activity of RIPK1 in necroptosis. (A) HT-22 cells were pretreated with or without 10 μM CDDO or 10 μM Nec-1s for30 min and then treated with 20 ng/mL human TNF-α (T), 20 nM SM-164 (S),and 50 μM zVAD.fmk (Z) for the indicated periods of time. The cell lysateswere analyzed by Western blotting using the indicated antibodies. (B) HT-22 cells were pretreated with 10 μM CDDO or 10 μM Nec-1s for 30 min andthen treated with T/S/Z for the indicated periods of time. The cell lysateswere analyzed by immunoprecipitation using anti-RIPK3 antibody and an-alyzed by Western blotting using the indicated antibodies. (C) HEK293T cellswere transfected with Flag-tagged RIPK1 expression plasmids for 24 h andthen treated with or without CDDO for 4 h. The cell lysates were analyzed byimmunoprecipitation (IP) using anti–p-S166–RIPK1 antibody and analyzed byWestern blotting using the indicated antibodies. (D) HT-22 cells were pre-treated with 10 μM CDDO for 30 min and then treated with the indicatedstimuli for 5 h. The cell lysates were analyzed by Western blotting using theindicated antibodies. C, CHX.

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factor-erythroid 2 p45-related factor 2 (Nrf2)–ARE system, andHSP90 inhibitor tanespimycin (17AAG) could present good in-hibitory effect on both necroptosis and ferroptosis (Fig. 4A).T0070907 (PPAR-γ antagonist), troglitazone (PPAR-γ agonist),TPCA-1 (IKK-β inhibitor), JAKi (Jak inhibitor), STAT3i (STATinhibitor), AV-412 (EGFR inhibitor), and MK2206 (Akt inhibitor)inhibited ferroptosis but not necroptosis (SI Appendix, Fig. S4A).Nrf2, a basic leucine zipper redox-sensitive transcription factor

and a master regulator of cellular antioxidant response, has beenshown to be a target for CDDO (42). However, in cells withNrf2 knockdown, we found that CDDO could still protect nec-roptosis and ferroptosis (Fig. 4 B and C). Since Nrf2 is a tran-scription factor, we used transcription and translation inhibitorsto block protein synthesis, and found that CDDO still protectedcell death induced by TNF-α/SM-164/zVAD.fmk (SI Appendix,Fig. S4B). We also evaluated Keap1, the cofactor of Nrf2, andfound that other Keap1 inhibitors such as sulforaphane (SFN)and dimethyl fumarate (DMF) inhibited necroptosis but notferroptosis (SI Appendix, Fig. S4C). Moreover, Keap1 inhibitorscannot protect necroptosis in HT-29 cells (SI Appendix, Fig. S4 Dand E). In addition, as Keap1 inhibition leads to the accumulation ofpolyubiquitylated Nrf2, we checked the Nrf2 induction effect ofthese compounds. In SI Appendix, Fig. S4F, we found that CDDO isnot a potent Nrf2 inducer compared with other Keap1 inhibitors.Thus, we conclude that Nrf2 is not the common regulatory nodalfor CDDO’s effect on necroptosis and ferroptosis.HSP90 is a highly evolutionarily conserved chaperone protein

and plays a critical role in the maturation, stability, and activa-tion of a number of diverse client proteins. Since HSP90 isknown to be associated with RIPK1 (29, 30), we next consideredthe role of HSP90 as the target of CDDO in blocking bothnecroptosis and ferroptosis. As shown in Fig. 4 D and E,knockdown of HSP90 in HT-22 cells inhibited necroptosis in-duced by TNF-α/SM-164/zVAD.fmk and TNF-α/CHX/zVAD.fmk and ferroptosis induced by glutamate and erastin. Thus,HSP90 is involved in mediating both necroptosis and ferroptosis.We next examined if CDDO could inhibit HSP90 in cells by

testing several biomarkers of HSP90 inhibition. Consistent withan inhibitor of HSP90, we found that CDDO induced remark-able increases in the levels of HSP70 and reduction in the levelsof a well-known HSP90 client protein, EGFR, in a dose-dependent

manner (Fig. 4F), which were both known to occur upon the in-hibition of HSP90 (43). Furthermore, we found that CDDO sig-nificantly disrupted the interactions between HSP90 and its clientproteins, including RIPK1 (Fig. 4G).These data suggest that CDDO inhibits HSP90 activity in cells

and HSP90 may be a common regulatory nodal for CDDO toblock both necroptosis and ferroptosis.

CDDO Inhibits RIPK1 Kinase Activity Through HSP90. A previousstudy reported that treatment with HSP90 inhibitors for longperiods of time (>10 h) reduced levels of RIPK1 (44). Whiletreatment with CDDO for long time (>10 h) also reduced thelevels of RIPK1 in HT-22 cells (SI Appendix, Fig. S5A), shortertreatment with CDDO (3–5 h), as used in our experimentalconditions, led to no obvious difference in the levels of RIPK1(Fig. 2). Thus, we next examined whether HSP90 could regulateRIPK1 kinase activity. We characterized the effects of twoHSP90 inhibitors, geldanamycin (GA) and 17AAG, in an RIPK1overexpression assay system. We found that the activation ofRIPK1 kinase, marked by p-S166, upon its overexpression in 293Tcells was dose-dependently inhibited by CDDO as well as by GA and17AAG, which were the most commonly used HSP90 inhibitors, whilethe levels of RIPK1 protein were not affected (Fig. 5A).To test if CDDO might inhibit RIPK1 through HSP90, we

constructed an HT-29 cell line that overexpressed HSP90. Consis-tent with HSP90 being a positive regulator of RIPK1 kinase activity,the cells overexpressing HSP90 showed enhanced RIPK1 kinaseactivity upon induction of necroptosis by TNF-α/SM-164/zVAD.fmk(Fig. 5B). Cotreatment of CDDO with TNF-α/SM-164/zVAD.fmkeffectively inhibited the activation of RIPK1 with or without over-expression of HSP90, suggesting that CDDO plays an inhibitory rolethrough HSP90 on RIPK1 kinase activity (Fig. 5B).Since HSP90 functions by promoting the structural stability of its

client proteins, inhibition of HSP90 may lead to their misfolding anddegradation (43). Consistently, we found that the treatment withCDDO reduced the protein levels of RIPK1 in the Nonidet P-40soluble fraction and increased its presence in the Nonidet P-40insoluble fraction (Fig. 5C), suggesting that inhibition of HSP90may lead to conformation changes in RIPK1 that lead to inhibitionof its kinase activity.

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Fig. 3. CDDO inhibits ferroptosis by blocking GPX4 degradation. (A) HT-22 cells were treated with the indicated stimuli for 6 h, followed by ROS mea-surement using the fluorescent probe carboxy-H2DCFDA. (B) HT-22 cells were treated with the indicated stimuli for 6 h. The lipid peroxidation was measuredby MDA assay. Ctrl, control. (C and E) HT-22 cells were treated with the indicated stimuli (10 μM CDDO, 10 μM erastin, 800 μM BSO) for the indicated periods oftime. The cell lysates were analyzed byWestern blotting using the indicated antibodies. (D) HT-22 cells were pretreated with 10, 15, or 20 μM CDDO for 30 minand then treated with BSO for 16 h. Cell viability was measured by CellTiterGlo assay. (F) HT-22 cells were transfected with a pool of GPX4-targeting siRNAsfor 12 h and then treated with or without 10 μM CDDO or 10 μM Fer-1 for 24 h. Cell viability was measured by CellTiterGlo assay. si-NC, negative control.Results shown are averages of triplicates ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

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As reported, treatment with TNF-α/SM-164 induces RIPK1-dependent apoptosis (RDA), which can be inhibited by Nec-1s(45). We then investigated the effect of CDDO on RDA. Treat-ment with CDDO also blocked the activation of RIPK1 kinaseinduced by treatment of TNF-α/SM-164 and TNF-α/5z7 (Fig. 5D).However, treatment with CDDO sensitized cells to RDA inducedby TNF-α/SM-164 or TNF-α/5z7 (SI Appendix, Figs. S1G andS5 B and C) and increased the cleavage of caspase-8 (Fig. 5D).Since inhibition of HSP90 is known to induce apoptosis (46) andRIPK1 is cleaved by caspase-8 (47), blocking the activation of RIPK1by HSP90 inhibitors is not sufficient to inhibit RDA.

Degradation of GPX4 by CMA Pathway During Ferroptosis. Degra-dation of GPX4 protein is a pivotal event in ferroptosis, which, inturn, promotes ROS and irreversible lipid peroxidation and,consequently, cell death (48). Since we found that CDDO blockedthe degradation of GPX4 in ferroptosis induced by glutamate orerastin, we next investigated the mechanism by which HSP90regulated the degradation of GPX4 using several protein deg-radation inhibitors for different types of protein degradation,including proteasome inhibitors (MG-132 and PS-341) and ly-sosome inhibitors [chloroquine (CQ), NH4Cl, and Baf-A1]. Wefound that only the lysosome pathway inhibitor CQ, NH4Cl, or

Baf-A1 could inhibit both cell death and GPX4 degradation in-duced by glutamate or erastin (Fig. 6 A and B).Since HSP90 regulates stability and activity of its client pro-

teins, we next used mass spectrometry to determine which clientprotein(s) of HSP90 might be involved in the degradation ofGPX4. We generated a line of 661W cells stably expressing Flag-GPX4. Using this cell line, we analyzed the binding proteins ofGPX4 by mass spectrometry. We found that among the proteinsbinding with GPX4, the levels of both HSC70 and HSP90 wereincreased after erastin treatment (Fig. 6C). The interactions ofHSC70 with HSP90 and GPX4 with HSC70 were confirmedusing coimmunoprecipitation (Fig. 6 D and E). Furthermore, wefound that the interaction of HSC70 and HSP90 was increased incells treated with erastin, which was reduced by CDDO (Fig. 6D).HSC70 is known to be associated with Lamp-2a to mediate

CMA (49). We next examined the effect of HSC70 and Lamp-2ain ferroptosis. We found that knockdown of Lamp-2a and HSC70significantly reduced ferroptosis and restored GPX4 protein levelsin cells treated with erastin (Fig. 6 F and G and SI Appendix, Fig.S6). As Lamp-2a and HSC70 are required for the protein degra-dation through CMA in the lysosome compartment, these resultsare consistent with lysosome inhibitors inhibiting the GPX4 degra-dation induced by glutamate or erastin.

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Fig. 4. CDDO targets HSP90 to inhibit both necroptosis and ferroptosis. (A) HT-22 cells were pretreated with the indicated inhibitors (25 μM t-BHQ, 1 μM17AAG, 10 μM CDDO) and then treated with TNF-α/zVAD.fmk (T/Z), T/SM-164 (S)/Z, or T/CHX (C)/Z (20 ng/mL T, 50 μM Z, 20 nM S, 1 μg/mL C) for 9 h orglutamate or erastin (50 mM glutamate, 10 μM erastin) for 12 h. Cell viability was measured by CellTiterGlo assay. (B–E) HT-22 cells were transfected with theindicated siRNA oligos for 48 h and then treated with the indicated stimuli (10 μM CDDO, 20 ng/mL T, 50 μM Z, 20 nM S, 50 mM glutamate, 10 μM erastin). Cellviability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies to determine the knockdownefficiency (si-HSP90α, mixture of two si-HSP90aa1 oligos; si-HSP90β, mixture of two si-HSP90ab1 oligos; si-HSP90mix, mixture of two si-HSP90aa1 and twosi-HSP90ab1 oligos; si-Nrf2-mix, mixture of four si-Nrf2 oligos; si-NC, negative control). (F) HT-22 cells were treated with 5, 10, or 15 μM CDDO for 10 h. The celllysates were analyzed by Western blotting using the indicated antibodies. (G) HT-29-HA-HSP90-α/β cells were treated with the indicated stimuli (10 μM CDDO,1 μM 17AAG, 10 μM Nec-1s) for 12 h. The cell lysates were analyzed by immunoprecipitation using anti-HA and analyzed by Western blot using the indicatedantibodies. Results shown are averages of triplicates ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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Overexpression of Lamp-2a can promote CMA (50). Consis-tently, we found that overexpression of Lamp-2a sensitized cellsto ferroptosis induced by glutamate and erastin (Fig. 6H). Con-sistent with the role of CMA in promoting the degradation ofGPX4, we found that the level of GPX4 protein in cells withoverexpressed Lamp-2a was lower than that of GFP-expressingcells upon erastin treatment (Fig. 6I).We next measured the effect of CMA on ferroptosis using a

photoactivatable (PA) reporter fused with a CMA-targeting motif(KFERQ-PA-mCherry) (51). Activation of CMA mobilizes thismCherry-tagged CMA substrate from the cytosol to lysosomes,which can be tracked as a change in the reporting fluorescence fromdiffuse signals to a punctate pattern. Treatment with erastin mark-edly increased the signals of this CMA reporter in HT1080 cells(Fig. 6J). Thus, CMA is activated in cells undergoing ferroptosis.

CDDO Protects Ferroptosis by Inhibiting CMA Pathway. HSC70 isknown to recognize and bind with substrates to deliver to thelysosome membrane, while Lamp-2a acts as a receptor totransport CMA substrates into the lysosome matrix for degra-dation (49, 50). We found that induction of ferroptosis by era-stin, glutamate, and RSL3 all led to increases in the levels ofLamp-2a in different cell lines (Fig. 7 A–C and SI Appendix, Fig.S7 A–C), and that the increased levels of Lamp-2a were inhibitedby the addition of CDDO (Fig. 7B and SI Appendix, Fig. S7 Aand B). HSP90 has been shown to associate with Lamp-2a at thelysosomal membrane and to regulate the functional dynamics ofthe Lamp-2a complexes for CMA activation (52). Consistently,the interaction between HSP90 and Lamp-2a was induced byerastin and reduced by treatment with CDDO, suggesting thaterastin may affect the interaction of HSP90 and Lamp-2a (Fig.7D). HSP90 may also regulate the stability of Lamp-2a, as the

treatment of CDDO also reduced the levels of Lamp-2a incontrol cells (SI Appendix, Fig. S7D).HSC70 and Lamp-2a interact with CMA substrates to promote

their degradation (52). We found that the interactions betweenGPX4 and HSC70 or Lamp-2a were markedly increased in theFlag-GPX4-661W cells after erastin treatment (Fig. 7C). CMAsubstrates are known to contain a KEFRQ-like motif that can berecognized and bound with HSC70 (53). Indeed, we found thatGPX4 harbored several pentapeptide sequences (124NVKFD128,169LIDKN173, and 187QVIEK191) that were consistent with HSC70recognition motifs. To functionally determine the importance ofthese motifs in GPX4 for its degradation during the ferroptosisprocess, we generated GPX4 mutants with a 2-aa mutation in thehypothetical CMA motifs (124NVKFD128 to 124AAKFD128,169LIDKN173 to 169LIDAA173, and 187QVIEK191 to 187AAIEK191).We found that GPX4 with mutated motifs 124NVKFD128 or187QVIEK191, which were marked as NVKFD* or QVIEK*, showedincreased resistance to erastin-induced degradation (Fig. 7E). Tofurther confirm the role of the KEFRQ-like motif, we generateda double-motif mutation. As shown in Fig. 7F, when both the124NVKFD128 and 187QVIEK191 motifs were mutated, degradationof this GPX4 double mutant was significantly impaired after erastintreatment. Moreover, this GPX4 double mutant failed to bind toHSC70 when overexpressed together in 293T cells (Fig. 7G).To further verify the degradation of GPX4 in the lysosomes, we

isolated lysosome-enriched fractions of cells treated with erastin. Aportion of GPX4 was associated with lysosomal factions under thecontrol condition. After treatment with erastin, the lysosomal frac-tions of HT-22 cells showed a marked enrichment of GPX4 and anincreased amount of Lamp-2a (Fig. 7H), demonstrating the lyso-somal targeting of GPX4 upon CMA activation.Since the increased levels of Lamp-2a are predicted to pro-

mote CMA (49, 50), we tested the possibility that ferroptosis maypromote the degradation of other CMA substrates. Indeed, wefound that the protein levels of GAPDH, one of the well-establishedCMA substrates (54), was also reduced in cells undergoing ferrop-tosis induced by erastin. Furthermore, the treatment with CDDOsignificantly restored the protein levels of GAPDH (Fig. 7I).Taken together, we conclude that activation of ferroptosis

leads to increases in the levels of Lamp-2a to promote CMA,which, in turn, mediate the degradation of multiple CMA sub-strates, including GPX4. Thus, the activation of CMA is involvedin mediating ferroptosis (Fig. 7J).

DiscussionOur study defines HSP90 as a common regulatory nodal in bothnecroptosis and ferroptosis (Fig. 7J). We show that HSP90 isimportant for mediating the activation of RIPK1 kinase in bothnecroptosis and RIPK1-mediated apoptosis (RDA). However,since inhibition of HSP90 by itself can strongly promote theactivation of apoptosis, inhibition of HSP90 by CDDO is notsufficient to block RDA. Furthermore, we show that activationof ferroptosis leads to increases in the levels of Lamp-2a andinduction of CMA in an HSP90-dependent manner, which, inturn, mediate the degradation of GPX4. Since CMA is involvedin mediating the degradation of oxidized proteins (54) and fer-roptosis triggers severe oxidative stress resulting in numerousoxidized and inactivated proteins (55), the oxidized environmentin cells undergoing ferroptosis may promote the activation ofCMA to mediate the degradation of many damaged proteins,including GPX4 and GAPDH, to mediate cell death. Our studysuggests that the activation of CMA is involved in the executionof ferroptosis.Necroptosis and ferroptosis have been defined as two in-

dependent forms of nonapoptotic cell death. Inhibition of thekey regulators in necroptosis such as RIPK1 and RIPK3 showedno impact on the process of ferroptosis (40). In addition, theferroptosis inhibitors Fer-1 and liproxstatin-1 cannot protect

Fig. 5. CDDO inhibits RIPK1 kinase activity through HSP90. (A) HEK293Tcells were transfected with Myc-tagged RIPK1 expression plasmids for 24 hand then treated with different concentrations of CDDO, GA, or 17AAG for4 h. The cell lysates were analyzed by Western blotting using the indicatedantibodies. (B) HT-29-HA-HSP90-α/β (HA-HSP90α/β) cells were treated withthe indicated stimuli [20 ng/mL TNF-α (T), 50 μM zVAD.fmk (Z), 20 nM SM-164(S), 10 μM CDDO] for 5 h. The cell lysates were analyzed by immunoprecip-itation (IP) using anti-HA and analyzed by Western blotting using the in-dicated antibodies. (C) HT-22 cells were treated with 10 μM CDDO for 1, 3, 5,or 7 h or with 1.5 μM 17AAG for 7 h and then harvested with 0.5% NonidetP-40 (NP-40) buffer. The soluble and insoluble fractions were separated bycentrifugation, and debris was dissolved with 1% SDS buffer. Both fractionswere subjected to SDS/PAGE, followed by immunoblotting analysis ofRIPK1 protein. (D) HT-22 cells were pretreated with 10 μM CDDO and thentreated with the indicated stimuli (20 ng/mL T, 20 nM S, 100 nM 5z7) for 5 h.The cells were harvested with 1% SDS buffer. The cell lysates were analyzedby Western blotting using the indicated antibodies.

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necroptosis (40). However, necroptosis and ferroptosis may oc-cur together to mediate pathology. Necrosis in the kidney is ahallmark of pathophysiological processes such as an acute is-chemic or toxic event during infection, renal transplant rejection,or thrombotic microangiopathies (56). Necroptosis and ferrop-tosis are both implicated in acute kidney injury. In animal modelsof ischemia reperfusion injury (IRI), deficiency of RIPK3 ortreatment with the necroptosis inhibitor Nec-1s was shown to beprotective of renal IRI by reducing cell necrosis and kidneydamage (57, 58). Inhibition of ferroptosis has additive benefits to

that of necroptosis in these acute kidney injury models (59, 60).Other experimental conditions [e.g., cell death induced by 1-methyl-4-phenylpyridinium in SH-SY5Y cells (61), hemoglobin or hemin incultured neurons (62), and BAY 87-2243 in melanoma cells (63)]present hallmarks of necroptosis and ferroptosis, which can be partiallyinhibited by Nec-1 and Fer-1. In addition, the loss of GPX4 triggersRIPK3-dependent necroptosis in mouse erythroid precursors (64).Thus, acute tissue injuries may involve both necroptosis andferroptosis. Our study suggests that HSP90 may represent a com-mon regulatory nodal between necroptosis and ferroptosis.

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Fig. 6. Degradation of GPX4 by the CMA pathway during ferroptosis. (A) HT-22 cells were pretreated with 25 μM CQ, 20 mM NH4Cl, 200 nM Baf A1, 10 μMMG-132, or 500 nM PS-341 for 1 h and then treated with 50 mM glutamate or 10 μM erastin for 12 h. Cell viability was measured by CellTiterGlo assay. Ctrl,control. (B) HT-22 cells were treated with 50 mM glutamate or 10 μM erastin for the indicated periods of time and then treated with CQ, NH4Cl, and Baf A1 for4 h before harvest. The cell lysates were analyzed by Western blotting using the indicated antibodies. (C) 661w-Flag-GPX4 cells were treated with 10 μMerastin for 18 h and harvested with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by massspectrometry. The abundances of binding proteins were normalized based on intensities of GPX4. (D) HT-1080-HA-HSP90-α/β (HA-HSP90α/β) cells were treatedwith the indicated stimuli (10 μM erastin, 10 μM CDDO) and then harvested with 0.5% Nonidet P-40 buffer. The cell lysates were analyzed by immuno-precipitation (IP) using anti-HA antibody and analyzed by Western blotting using the indicated antibodies. (E) HEK293T cells were cotransfected with HA-tagged GPX4, HA-tagged GPX4-U46C (Sec/Cys GPX4 mutant), and Flag-tagged HSC70 expression plasmids as indicated for 24 h and then lysed with 0.5%Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting using the indicated antibodies. (F andG) HT-22 cells were transfected with the indicated siRNA oligos for 48 h and then treated with the indicated stimuli (50 mM glutamate or 10 μM erastin). Cellviability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies (si-HSC70, mixture of three si-HSC70 oligos; si-Lamp-2a, mixture of two si-Lamp-2a oligos; si-NC, negative control). (H and I) HT-22 cells with or without overexpressed Lamp-2a were treatedwith glutamate or erastin (50 mM glutamate, 10 μM erastin) for 12 h. Cell viability was measured by CellTiterGlo assay. The cell lysates were analyzed byWestern blotting using the indicated antibodies. (J) HT-1080 cells stably expressing KFERQ-PA-mCherry1 were photoactivated by 405-nm light and thentreated with serum starvation for 24 h or with erastin for 15 h or 18 h. The images were analyzed for quantification by ImageJ software. (Scale bars: 25 μm.)Results shown are averages of triplicates ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

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Our study provides evidence for the involvement of HSP90, aubiquitous heat shock protein, in mediating ferroptosis re-sistance. Although HSP90 may not bind directly with GPX4, wefound that HSP90 participated in the ferroptosis process byregulating the levels of Lamp-2a in the CMA pathway. The heatshock response is an important system for maintaining cellularhomeostasis when cells are under stress, such as in the oxida-tive stress environment during ferroptosis. Activation of theHSF1/HSPB1 pathway by heat shock or overexpression nega-

tively regulated erastin-induced ferroptosis (65). Likewise, heatshock triggered an iron-dependent cell death pathway in plantscharacterized by depletion of GSH and ascorbic acid and accu-mulation of cytosolic and lipid ROS, indicative of ferroptosis (66).HSP90 has been recognized as a promising target for a num-

ber of diseases associated with aberrant protein signaling, in-cluding cancer, neurodegeneration, and infectious diseases (67–70). Multiple HSP90 inhibitors have been developed and haveadvanced into human clinical trials. The role of HSP90 in both

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Fig. 7. CDDO protects ferroptosis by inhibiting the CMA pathway. (A) HT-22 cells were treated with 10 μM erastin for the indicated periods of time and thenlysed with 1% SDS buffer. The cell lysates were analyzed by Western blotting using the indicated antibodies. (B) 661W cells were pretreated with 10 μM CDDOfor 30 min and then treated with 10 μM erastin for 12 or 24 h. The cell lysates were analyzed by Western blotting using the indicated antibodies. (C) 661W-Flag-GPX4 cells were treated with 10 μM erastin for the indicated periods of time. The cell lysates were subjected to immunoprecipitation (IP) using anti-Flagantibody and then analyzed by Western blotting using the indicated antibodies. (D) HT-1080 cells stably expressing HA-HSP90-α/β (HA-HSP90α/β) were treatedwith the indicated stimuli for 12 h. The cell lysates were analyzed by immunoprecipitation using anti-HA and analyzed by Western blotting using the in-dicated antibodies. (E and F) 661W cells stably expressing Flag-GPX4 or the indicated Flag-GPX4 mutants were treated with 10 μM erastin for the indicatedperiods of time. The cell lysates were analyzed by Western blotting using the indicated antibodies. (G) HEK293T cells were cotransfected with Flag-taggedGPX4, Flag-tagged GPX4-Q*N*, and HA-tagged HSC70 expression plasmids as indicated for 24 h and then lysed with 0.5% Nonidet P-40 buffer. The cell lysateswere immunoprecipitated with anti-HA antibody, followed by Western blotting using the indicated antibodies. (H) HT-22 cells were treated with 10 μMerastin for 18 h and then subjected to subcellular fractions isolation, followed by Western blotting, using the indicated antibodies. (I) HT-22 cells werepretreated with 10 μM CDDO for 30 min and then treated with 10 μM erastin for the indicated periods of time. The cell lysates were analyzed by Westernblotting using the indicated antibodies. (J) Graphic summary for the role of HSP90 in both necroptosis and ferroptosis. HSP90 regulates the activation ofRIPK1 kinase activity to control the necroptosis pathway. In the ferroptosis pathway, HSP90 regulates the degradation of GPX4 through CMA. Treatment witherastin or glutamate promotes lipid peroxidation and activation of CMA, which mediates the degradation of GPX4 and other CMA substrates to promoteferroptosis.

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necroptosis and ferroptosis presents a perspective for drug dis-covery of diseases involving necroptosis or ferroptosis.

Materials and MethodsReagents and Antibodies. Recombinant human TNF-α (catalog no. C008) waspurchased from Novoprotein Scientific, Inc. The 17AAG, GA, and erastinwere purchased from TargetMol. MG-132 and Fer-1 were purchased fromSelleckchem. Carboxy-H2DCFDA and 5Z-7-oxozeaenol were purchased fromSigma–Aldrich. CDDO, TPCA-1, t-BHQ, MK2206, AV-412, T0070907, andtroglitazone were purchased from MedChemExpress. Nec-1s, zVAD.fmk, andSM-164 were custom-synthesized. The following commercial antibodieswere used in this study: RIPK1 (3493; Cell Signaling Technology); p-S166–RIPK1 (Biolynx); p-S345–MLKL (ab196436; Abcam); p-S358–MLKL (91689;Cell Signaling Technology); TNFR1 (AF-425-PB; R&D Systems); Myc (C3956;Sigma–Aldrich); GPX4 (ab125066; Abcam); HSP90-α (13171-1-AP; Proteintech);HSP90-β (ADI-SPA-845-F; Enzolife); Lamp-2a (51-2200; Invitrogen); HSC70(10654-1-AP; Proteintech); and p-T231/S232–RIPK3, IκBα, p-IκBα, IKK-α, IKK-β,cl-caspase-8, and Nrf2 (Cell Signaling Technology). All of the siRNA oligoswere ordered from GenePharma.

Cell Culture. HT-22, 661W, HT-1080, and HEK293T cells were cultured inDMEM (Gibco) with 10% (vol/vol) FBS (Gibco) and 100 units/mL penicillin/streptomycin. Jurkat cells were cultured in RPMI-1640 (Gibco) with 10% (vol/vol)FBS (Gibco) and 100units/mLpenicillin/streptomycin.HT-29 cellswere cultured inMcCoy’s 5A (Gibco) with 10% (vol/vol) FBS (Gibco) and 100 units/mL penicillin/streptomycin. All cells were cultured at 37 °C with 5% CO2.

CellTiter-Glo Assay and PI Staining for Cell Survival. The CellTiter-Glo lumi-nescent cell viability kit was from Promega. The viability of cells was nor-malized to that of control cells treated with vehicle alone. For the PI staining,1 μM PI and 10 μg/mL Hoechst stain were added to cells for 10 min at roomtemperature and imaged using a Leica fluorescence microscope. All cellsurvival assays were performed in triplicate.

Immunoprecipitation. Cells were lysed with Nonidet P-40 buffer [150 mMNaCl, 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40,5% glycerol] supplemented with 1 mM PMSF, 1× protease inhibitor mixture(Roche), 10 mM β-glycerophosphate, 5 mMNaF, and 1 mMNa3VO4. Debris wasprecipitated, and the lysate was incubated with an antibody overnight at 4 °C.The immunocomplex was captured by Protein A/G Agarose (Life Technologies)with the appropriate antibodies for 3–5 h at 4 °C. Beads were washed fourtimes, and the immunocomplex was eluted from beads by loading buffer.

In Vitro Kinase Assays. HEK293T cells were transfected with Flag-taggedRIPK1 expression plasmids for 24 h and immunoprecipitated with anti-Flagantibody. The immunoprecipitated complexes were divided into equalparts and incubated in 30 μL of kinase assay buffer [20 mM Hepes (pH 7.5),2 mM DTT, 10 mM MgCl2] supplemented with 50 μM cold ATP for 30 min at30 °C in the presence of different compounds. The reaction was stopped byadding SDS/PAGE loading buffer and heating at 95 °C for 5 min.

MDA Assay. The relative MDA concentration in cell lysates was assessed usingan MDA Assay Kit (Beyotime) according to the manufacturer’s instructions.

DPPH Assay. The stable radical DPPH was dissolved in methanol to a finalworking concentration of 0.05mM. Onemilliliter of DPPH solutionwas addedto a small volume (<5 μL) of each test compound dissolved in DMSO. Thefinal concentration of each test compound was 0.05 mM. Samples wereinverted several times and allowed to incubate at room temperature for30 min. Samples were then aliquoted to white, 96-well, solid-bottomeddishes (Corning), and the absorbance at 517 nm was recorded. All valueswere normalized to background (methanol only).

KFERQ-PA-mCherry1 Reporter Assay. The pLVX-KFERQ-PA-mCherryN1 plasmidwas a gift from Guoqiang Chen and Qian Zhao of Shanghai Jiao TongUniversity School ofMedicine, Shanghai, China (51). HT-1080 cells were stablyinfected with pLVX-KFERQ-PA-mCherryN1 lentivirus. For CMA activity anal-ysis, cells were photoactivated by a 405/20-nm LED array, followed bytreatment, and were then fixed and costained with DAPI. The images wereacquired using a Leica SP8 confocal microscope. Activation of CMA activitywas calculated as the number of cells that present bright puncta under a 20×or 63× objective and were quantified using ImageJ software (NIH).

Lysosome Isolation. Lysosome fractions of cultured cells were obtained usinghomogenization and sequential centrifugation. Lysosomes were isolatedusing a Lysosome Enrichment Kit (Pierce) according to the kit protocol.

Mass Spectrometry. The 661w-Flag-GPX4 cells were treated with 10 μMerastin for 18 h and harvested with 0.5% Nonidet P-40 buffer. The cell ly-sates were immunoprecipitated with anti-Flag antibody. The proteins weretrypsin-digested and analyzed on an Orbitrap Q Exactive mass spectrometer(Thermo Scientific). The protein identification and label-free quantificationwere done using MaxQuant software. The tandem mass spectra weresearched against the UniProt mouse protein database and a set of com-monly observed contaminants. The precursor mass tolerance was set as20 ppm, and the fragment mass tolerance was set as 0.1 Da. The cysteinecarbamidomethylation was set as a static modification, and the methionineoxidation was set as a variable modification. The false discovery rates at thepeptide spectrum match level and protein level were controlled below 1%.

Statistics. Data are expressed as the mean ± SEM. Error bars indicate the SEM.Pairwise comparisons between two groups were performed using the Stu-dent’s t test. Differences were considered statistically significant if *P < 0.05,**P < 0.01, ***P < 0.001, or ****P < 0.0001, or as not significant. At leastthree independent biological repeats were included in each data point. Eachexperiment was repeated at least three times.

ACKNOWLEDGMENTS. We thank Drs. Guoqiang Chen and Qian Zhao(Shanghai Jiao Tong University School of Medicine) for pLVX-KFERQ-PA-mCherryN1 plasmid. This work was supported by the National Key R&D Pro-gram of China (Grant 2016YFA0501900) and the China National NaturalScience Foundation (Grants 31530041 and XDPB10).

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