1. Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death
Scott J. Dixon, Kathryn M. Lemberg, Michael R. Lamprecht, Rachid Skouta, Eleina M. Zaitsev, Caroline E. Gleason, Darpan N. Patel, Andras J. Bauer, Alexandra M. Cantley, Wan Seok Yang, Barclay Morrison, Brent R. Stockwell 
Cell 
Volume 149, Issue 5, Pages (May 2012)
DOI: /j.cell
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2. Figure 1
Erastin-Induced Death Triggers the Accumulation of Cytosolic ROS, Whose Production Can Be Inhibited by DFO
(A) Visualization of HT-1080 cell viability over time ±erastin (Era, 10 μM) and deferoxamine (DFO, 100 μM).
(B and C) Cytosolic and lipid ROS production assessed over time (2, 4, and 6 hr) by flow cytometry using H2DCFDA and C11-BODIPY.
(D) Mitochondrial ROS assessed in HT-1080 cells treated for 6 hr with erastin ±DFO, as above, or with rotenone (250 nM) ±DFO. In (A)–(D), representative data from one of four experiments are shown.
(E) Erastin-induced death in 143B ρ0 and ρ+ cells.
(F) mtDNA-encoded transcript levels in ρ0 and ρ+ cells. Results in (E) and (F) are mean ±SD from one of three representative experiments.
See Figure S1 for additional data showing that iron-dependent cell death occurs independently of the mitochondrial electron transport chain.
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3. Figure 2 Erastin-Induced Oxidative Death Is Iron Dependent
(A) Transmission electron microscopy of BJeLR cells treated with DMSO (10 hr), erastin (37 μM, 10 hr), staurosporine (STS, 0.75 μM, 8 hr), H2O2 (16 mM, 1 hr), and rapamycin (Rap, 100 nM, 24 hr). Single white arrowheads, shrunken mitochondria; paired white arrowheads, chromatin condensation; black arrowheads, cytoplasmic and organelle swelling and plasma membrane rupture; black arrow, formation of double-membrane vesicles. A minimum of 10 cells per treatment condition were examined.
(B) Normalized ATP levels in HT-1080 and BJeLR cells treated as in (A) with the indicated compounds. Representative data (mean ±SD) from one of three independent experiments are shown.
(C) Modulatory profiling of known small-molecule cell death inhibitors in HT-1080, BJeLR, and Calu-1 cells treated with erastin (10 μM, 24 hr).
(D) Effect of inhibitors on H2DCFDA-sensitive ROS production in HT-1080 cells treated for 4 hr.
(E) Modulatory profiling of ciclopirox olamine (CPX), DFO, ebselen (Ebs), trolox (Tlx), U0126, and CHX on oxidative and nonoxidative lethal agents.
See Figure S2 for related data showing that iron-dependent cell death is independent of proapoptotic proteins.
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4. Figure 3 Erastin-Induced Ferroptosis Exhibits a Unique Genetic Profile
(A) Outline of the shRNA screen and confirmation pipeline.
(B and C) Six high-confidence genes required for erastin-induced ferroptosis. (B) Viability of HT-1080 cells infected with shRNAs for 72 hr and treated with erastin (10 μM, 24 hr). (C) mRNA levels for hairpins shown in (B) determined by using RT-qPCR. Data in (B) and (C) are mean ±SD from one of three experiments.
(D and E) Effect of shRNA-mediated silencing of high-confidence genes by using the best hairpin identified by mRNA silencing efficiency in (C) on cell viability. (D) Viability of various cell lines treated with a lethal dose of erastin (indicated in brackets) for 24 hr. (E) Viability of HT-1080 cells treated with various death-inducing or cytostatic compounds. For (D) and (E), % rescue was computed relative to each shRNA alone +DMSO.
(F) Cartoon outline of glutamine (Gln) metabolism. Red box indicates mitochondria.
(G) Images of HT-1080 cells treated with aminooxyacetic acid (AOA) ±dimethyl α-ketoglutarate (DMK) ±erastin.
See also Figure S3.
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5. Figure 4 Identification and Characterization of Ferrostatin-1
(A) Structure of ferrostatin-1 (Fer-1). The molecular weight (MW) is indicated.
(B) Effect of resynthesized Fer-1 (0.5 μM) on the lethality of various compounds in HT-1080 cells. All drug treatments were for 24 hr.
(C) Effect of Fer-1 and U0126 on ERK phosphorylation in HT-1080 cells.
(D) Effect of DFO, CHX, trolox (Tlx), and Fer-1 on HT-1080 cell proliferation over 48 hr as assessed by Vi-Cell.
(E) Effect of Fer-1 (0.5 μM) on erastin (10 μM)-induced ROS production in HT-1080 cells (4 hr treatment).
(F) Cell-free antioxidant potential monitored by changes is the absorbance at 517 nm of the stable radical DPPH.
(G) Dose-response relationship for inhibition of erastin (10 μM, 24 hr)-induced death in HT-1080 cells by Fer-1 and analogs.
(H) Chemical structure of various Fer-1 analogs tested in (F) and (G).
(I) Correlation between predicted partition coefficient (log P) and the ability of various Fer-1 analogs to prevent erastin-induced death.
(J) Dose-response relationship for inhibition of erastin (10 μM, 24 hr)-induced death by various antioxidants.
(K) Plot of predicted partition coefficient (log P) and ability of various antioxidants to prevent erastin-induced death. Data in (B), (D), (F), (G), and (J) represent mean ±SD from one of three representative experiments.
For additional data on Fer-1 identification and characterization, see also Figure S4.
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6. Figure 5
Effects of Fer-1 on Excitotoxic Cell Death in Organotypic Hippocampal Slice Cultures
(A) Cartoon outline of hippocampal slice procedure.
(B) Bright-field and fluorescent images of PI staining of treated hippocampal slices. Slices were treated with glutamate (5 mM, 3 hr) ±Fer-1 (2 μM), CPX (5 μM), or MK-801 (10 μM). Representative images from 1 of 6 slices per condition are shown.
(C–E) Quantification of the effects depicted in (B). Data shown are mean ±SD. Data were analyzed using a two-way ANOVA (brain region x drug treatment) followed by Bonferroni posttests.
∗∗p < 0.01 and ∗∗∗p <
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7. Figure 6 Erastin Inhibits the Activity of System xc−
(A) Modulatory profile of HT-1080 cells treated with different lethal compounds and inhibitors.
(B) Cartoon depicting the composition and function of system L and system xc−. Cys, cystine; NAA, neutral amino acids.
(C) SLC7A11 mRNA levels in compound-treated (6 hr) HT-1080 cells determined by RT-qPCR.
(D and E) Effect of silencing SLC7A11 by using siRNA on erastin (10 μM, 8 hr) induced death (D) and mRNA levels (E) in HT-1080 cells.
(F) Normalized Na+-independent [14C]-cystine uptake by HT-1080 cells in response to various drugs. Data are represented as mean ±SD, n = 3.
(G) Identification of SLC7A5 as the lone target identified by erastin affinity purification in both BJeH and BJeLR cells.
(H) Metabolic profiling of system L and nonsystem L substrate amino acid levels in erastin-treated Jurkat cells.
(I) Effect of L-glutamic acid (L-Glu, 12.5 mM) and D-phenylalanine (D-Phe, 12.5 mM) on erastin-induced death in HT-1080 cells.
See also Figure S5.
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8. Figure 7 Role of NOX in Erastin-Induced Death
(A) Outline of NOX pathway. Inhibitors are shown in green. PPP, pentose phosphate pathway.
(B) Effect of NOX pathway inhibitors on erastin-induced death in Calu-1 and HT-1080 cells. GKT, GKT
(C and D) Effect of shRNA silencing of the PPP enzymes glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate dehydrogenase (PGD) on viability of erastin (2.5 μM)-treated Calu-1 cells. Infection with shRNA targeting VDAC2 was used as a positive control. Relative mRNA levels in (D) were assessed by RT-qPCR following shRNA knockdown. Data in (B), (C), and (D) represent mean ±SD.
(E) Model of ferroptosis pathway. The core ferroptotic lethal mechanism is highlighted in blue.
See Figure S6 for additional data supporting a role for the PPP/NOX pathway in erastin-induced cell death.
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9. Figure S1
RSLs Trigger Iron-Dependent Cell Death Independent of the Mitochondrial Electron Transport Chain, Related to Figure 1
(A) Viability of HT-1080 cells treated with erastin ±ferric ammonium citrate (FAC) over time as assessed in triplicate by Vi-Cell.
(B) Viability of HT-1080 cells treated with DMSO or erastin ±FAC, ferric citrate (FC), iron chloride hexahydrate (IHC), manganese chloride (Mn), nickel sulfate hexahydrate (Ni), cobalt chloride hexahydrate (Co) or copper sulfate (Cu). FAC was used at 10 μg/ml, all other metals were used at 25 μM. Cell viability was assessed by Trypan Blue exclusion (Vi-Cell) in triplicate and the effects of the erastin+metal combination were expressed as a percentage of the DMSO+metal viability alone.
(C and D) Mitochondrial superoxide levels in 143B cells assessed by flow cytometry using MitoSOX. Treatments used: 250 nM rotenone (Rote, R), 100 μM DFO alone or in combination, as indicated.
(E) Viability of 143B ρ+ and ρ0 cells treated for 24 hr with RSL3 and assessed by Alamar Blue.
All experiments were repeated two to four times with similar results and representative data from one experiment are shown. Data in panels A, B, and E represent mean ±SD from multiple replicates within one experiment.
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10. Figure S2
Ferroptosis Occurs in Mouse Embryonic Fibroblasts, Is Independent of Bax and Bak, and Can Be Attenuated by the Late Addition of Inhibitors, Related to Figure 2
(A) SV40-transformed MEFs (control and Bax/Bak double knockout, DKO) were treated with erastin ±DFO, Trolox, U0126 or cycloheximide (CHX) for 24 hr as indicated.
(B) Wild-type and DKO MEFs were treated with staurosporine (STS) for 24 hr at the indicated concentrations to induce apoptosis. Bax/Bak double knockout MEFs are more resistant to STS, as expected. In (A and B), cell viability was assessed by Alamar Blue. Experiments were repeated twice with similar results and representative data from one experiment are shown. All values are mean ±SD from multiple replicates within each experiment.
(C) Cells were treated ±erastin (10 μM) and cotreated with the indicated inhibitors. Inhibitors were added either at the same time as erastin (0 hr) or 2–6 hr later (+2, +4, and +6 hr). 2,2-bipyridyl (2,2-BP) is a membrane permeable iron chelator.
All cells were photographed 24 hr after the start of the experiment. This experiment was repeated three times with similar results and representative data from one experiment are shown.
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11. Figure S3 IREB2 Is Essential for Ferroptosis, Related to Figure 3
(A–C) HT-1080 cells were infected with shRNAs targeting IREB2 and FBXL5 for 3 days then examined for gene expression or drug sensitivity. (A,B) Reciprocal transcriptional regulation of iron-regulated genes induced by silencing of IREB2 and FBXL5 as assessed by RT-qPCR. DFO treatment (48 hr) was used as a control for changes in gene expression. ISCU, FTH1, FTL and TFRC are known iron-regulated genes (Sanchez et al., 2011). (C) Silencing of FBXL5 sensitizes to erastin-induced death.
(D) Aminooxyacetic acid (AOA), but not dichloroacetic acid (DCA), inhibits erastin-induced death in HT-1080 and BJeLR cells.
All data are mean ±SD from multiple replicates within one experiment. All experiments were performed 2–4 times with similar results. Representative data from one experiment are shown.
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12. Figure S4
Identification of Fer-1 and Fer-1 Structure-Activity Relationship Analysis, Related to Figure 4
(A) Retesting in 10-point, 2-fold dilution series of the top four compounds validated to suppress erastin-induced death in HT-1080 cells.
(B) The chemical structures of the top 4 compounds.
(C) Effect of varying the Fer-1 structure on the ability of such compounds to inhibit death in erastin (10 μM)-treated HT-1080 cells. Cell viability was assessed by Alamar Blue in quadruplicate. EC50 values (nM) were computed from dose response curves using Prism. Log P (Slog P) values were computed using Molecular Operating Environment.
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13. Figure S5
Analyzing the Role of Calcium and System xc− in Ferroptosis, Related to Figure 6
(A) Viability of HT-1080 cells treated for 24 hr with erastin +/− DMSO, the calcium chelators BAPTA-AM or Fura-2, or, as a positive control for death rescue, the iron chelator ciclopirox olamine (CPX), at the indicated concentration.
(B) The viability of HT-1080 cells treated for 24 hr with erastin, monosodium L-glutamic acid or RSL3 ±inhibitors was assessed using Alamar Blue. For (A–C), values represent mean ±SD from multiple replicates within one experiment. The entire experiment was repeated twice and representative data from one experiment are shown.
(C) Sulfasalazine, like erastin, displays RAS-selective lethal properties in the BJ cell series assay.
(D) HT-1080 cells were transfected for 48 hr with either a control plasmid (pMaxGFP) or pCMV6-SLC7A11-DDK then treated with DMSO, erastin or SAS, as indicated, and photographed.
(E) [14C]-cystine uptake into HT-1080 cells was measured under Na+-free conditions in response to DMSO, erastin, and RSL3.
Data represent mean ±SD, n = 3.
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14. Figure S6
Erastin-Induced Death Is Prevented by Inhibition of the PPP/NOX Pathway, Related to Figure 7
(A) Relative expression of NOX family catalytic subunit mRNAs in Calu-1 cells assessed by RT-qPCR.
(B) The viability of Calu-1 and BJeLR cells in response to erastin (10 μM) ±the PPP inhibitor 6-aminonicotinamde (6-AN, 200 μM) was assayed after 24 hr by Vi-Cell. Data in (A) and (B) represent mean ±SD of replicates from one experiment.
(C) H2DCFDA-reactive ROS were measured in BJeLR cells treated for 8.5 hr, as indicated, prior to the onset of overt death in these cells.
Experiments were performed three times with similar results and representative data from one experiment are shown.
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15. Cell  , DOI: ( /j.cell )
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16. Cell  , DOI: ( /j.cell )
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