Menaquinone-4 (V9378), phylloquinone (V3501), menadione (M5625), MK4 epoxide (75618), vitamin K3 epoxide (51455), α-TOC (T3251), warfarin (A2250), warfarin sodium (PHR1435), dicoumarol (M1390), l-buthionine sulfoximine (BSO; B2515) N-acetyl-l-cysteine (A7250), lipopolysaccharide (LPS; L2880) and MCC950 (5381200001) were purchased from Sigma-Aldrich. Ferrostatin-1 (Fer1; 17729), RSL3 (19288), FINO2 (25096), ML162 (20455), ML160 (23282) and staurosporine (81590) were purchased from Cayman. The following chemicals were obtained as indicated: erastin (329600, Merck Millipore), 17-AAG (A10010, Adqoo), l-glutamate (16911-22, Nacalai tesque), menadiol (M323135, TRC), iFSP1 (8009-2626, ChemDiv), liproxstatin-1 (Lip1, S7699, Selleckchem), BV-6 (S7597, Selleckchem), Trolox (56510, Fluka), recombinant mouse TNF (PMC3014, Thermo Fishier), nigericin (N1495, Thermo Fisher), zVAD-FMK (ALX-260-02, Enzo Life Sciences) and Nec 1s (2263, BioVision).
4-OH-TAM-inducible Gpx4−/− mouse immortalized fibroblasts (Pfa1) were described previously8. HT-1080 (CCL-121), 786-O (CRL-1932), A375 (CRL-1619), B16F10 (CRL-6475), H9C2 (CRL-1446), NRK49F (CRL-1570), C2C12 (CRL-1772), HepG2 (HB-8065), Jurkat (TIB-152), L929 (CCL-1) and HEK293T (CRL-3216) cells were obtained from ATCC. Panc-1 cells were obtained from Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). THP-1 cells were obtained from DSMZ (Germany). HT-22 cells were purchased from Millipore. All cell lines, except Jurkat and THP-1, were maintained in DMEM high glucose (4.5 g l−1 glucose, 21969-035, Gibco) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 1% penicillin/streptomycin at 37 °C with 5% CO2, unless stated otherwise. Jurkat and THP-1 cells were maintained in RPMI 1640 Glutamax medium (61870010, Gibco) supplemented with 10% heat-inactivated FBS. GPX4-KO cells were maintained in medium containing Lip1 (1 μM) to prevent ferroptosis. All cells were regularly tested for mycoplasma contamination.
Cell viability assays
Cells were seeded on 96-well plates and allowed to adhere overnight. On the next day, cells were treated with cell death inducers. Vitamin K compounds were added to the medium 1 h prior to the treatment with the ferroptosis inducing agents. Warfarin, iFSP1 and dicoumarol were added during cell seeding on 96-well plates. Cell viability was assessed 24 h (RSL3, erastin, FINO2, ML210, ML162, glutamate and staurosporine), 48 h (17AAG and FIN56) and 60 h (BSO) after the treatment using AquaBluer (MultiTarget Pharmaceuticals) as an indicator of viable cells. The cell viability was expressed as relative values compared to the control sample, which was defined as 100%. Pfa1 cells were seeded on 96-well plates (500 cells per well) and treated in a dilution series of the compounds and 1 μM 4-OH-TAM to induce the KO of Gpx4. Cell viability of Pfa1 cells was assessed 72 h after TAM treatment. To directly monitor cell death, LDH release was used, whereby LDH activity in medium was measured using LDH Cytotoxity Detection kit (Takara Bio). For induction of ferroptosis in HT-1080 cells (seeded on 96-well plates at 2,000 cells per well), RSL3 (0.3 µM), erastin (2 µM), ML210 (1 µM), ML162 (1 µM), FIN56 (1 µM), BSO (500 µM) and FINO2 (5 µM) were used. For induction of ferroptosis in 786-O cells by RSL3, 3,000 cells per well were seeded in 96-well plates. For cell viability assay of A375 GPX4-KO cells and 786-O GPX4-KO cells, 500 cells per well were plated in 96-well plates and incubated without Lip1. For cell viability assay of H9C2 cells, 4,000 cells per well were plated in 96-well plates with low-glucose DMEM (1.0 g l−1 glucose, Gibco) supplemented with 1% FBS to enhance susceptibility to ferroptosis27. For apoptosis induction, Jurkat cells (20,000 cells per well) were incubated with soluble human Fas ligand (30 ng ml−1, ALX-522-020, Enzo Life Sciences) for 24 h, and Pfa1 cells (1,500 cells per well) were co-incubated with mouse TNF (10 ng ml−1) and BV-6 (400 nM) for 24 h. For necroptosis induction, L929 cells (10,000 cells per well) were co-incubated with mouse TNF (10 ng ml−1), BV-6 (400 nM) and zVAD-FMK (30 μM) for 24 h. For pyroptosis induction, LPS (1 μg ml−1, 4h)-stimulated THP-1 cells (20,000 cells per well) were pretreated with vitamin K or MCC950 for 1 h and then incubated with nigericin (10 μM) for 2 h.
Preparation of lentiviral particles
Lentiviral packaging system consisting of a transfer plasmid, psPAX2 (12260, Addgene), and pMD2.G (12259, Addgene) was co-lipofected into HEK293T cells using the X-tremeGENE HP agent (Roche). Cell culture supernatants containing viral particles were collected 48 h after the transfection and used to transduce the cell line of interest after filtration using a 0.45 μm low protein binding syringe filter.
CRISPR–Cas9-mediated gene knockout
Single guide RNAs (sgRNA) were designed to target critical exons of the genes of interest to be inactivated as listed in Supplementary Table 1. The guides were cloned in the BsmBI-digested lentiCRISPR v2-blast and lentiCRISPR v2-puro vectors (98293 and 98290, Addgene), or BbsI-digested pKLV-U6gRNA(BbsI)-PGKpuro2aBFP vector (50946, Addgene).
Transient expression of the CRISPR–Cas9 system
A375 and B16F10 cells were transiently co-transfected with the desired sgRNA expressing lentiCRISPR v2-blast and lentiCRISPR v2-puro using the X-tremeGENE HP agent (Roche). One day after transfection, cells were selected by incubation with puromycin (1 µg ml−1) and blasticidin (10 µg ml−1). After selection, single-cell clones were picked and knockout clones were identified by sequencing out-of-frame mutations and immunoblotting.
Stable expression of CRISPR–Cas9 system
786-O and HepG2 cells were infected with VSV-G pseudotyped lentiviral particles containing the transfer plasmid of lentiCRISPRv2-puro and lentiCRISPRv2-blast. One day after transfection, cells were treated with selection antibiotics (blasticidin 15 µg ml−1 and puromycin 1.5 µg ml−1 for 786-O, and puromycin 1 µg ml−1 for HepG2). After the selection, loss of FSP1 expression in HepG2 cells was confirmed by immunoblotting of batch cultures. Regarding 786-O GPX4-KO cells, single-cell clones were picked and individually expanded. KO clones were identified by immunoblotting.
Doxycycline-inducible Cas9 expression system
Dox-inducible Cas9 expressing cells were generated by transducing 786-O and HT-1080 cells with VSV-G coated ecotropic lentiviral particles containing pCW-Cas9-Blast (83481, Addgene). After blasticidin selection (15 and 10 µg ml−1 for 786-O and HT-1080, respectively), single-cell clones expressing Dox-inducible Cas9 were identified by immunoblotting. pCW-Cas9-Blast expressing 786-O and HT-1080 cells were used to generate FSP1-KO cells by lentiviral infection with particles containing the desired sgRNA expressing pKLV-U6gRNA(sgRNA)-PGKpuro2ABFP. One day after infection, cells were selected with puromycin (1.5 and 1 µg ml−1 for 786-O and HT-1080, respectively), and then incubated with doxycycline (Dox) (10 µg ml−1) for 5 days to express Cas9. After Cas9 induction, loss of FSP1 expression in the cell pool was confirmed by immunoblotting.
Overexpression and Dox-inducible expression of FSP1
Codon-optimized human FSP1 gene with a C-terminal HA tag was cloned in the expression vector p442-Neo and Dox-inducible lentivirus vector pSLIK-Neo (25735, Addgene). FSP1-KO cells were infected with VSV-G pseudotyped lentiviral particles containing the hFSP1-cloned transfer plasmids. One day after infection, cells were selected with geneticin (1 mg ml−1). Reconstitution of FSP1 expression was verified by immunoblotting. Dox-inducible FSP1 expression was additionally verified by immunoblotting after treatment with increased concentrations of Dox for 24 h. For determining cell viability, cells were treated with increasing concentrations of Dox overnight and maintained in medium containing the same concentration of Dox during the assay period.
HT-1080 cells (80,000 cells) were seeded on µ-Dish 35 mm low (80136, i-bidi) and incubated overnight. On the next day, the cells were treated with or without 3 μM MK4 1 h before the addition of 0.5 μM RSL3. Live-cell imaging was performed using 3D Cell Explorer and Eve software v1.8.2 (Nanolive). Images were obtained at 1 min intervals. During imaging, the cells were maintained at 37 °C and 5% CO2 by using a temperature-controlled incubation chamber.
BODIPY 581/591 C11 staining
Pfa1 cells (50,000 cells per well) were seeded on 6-well dishes one day prior to the experiment. On the next day, cells were treated with 0.3 μM RSL3. Phylloquinone (3 µM), MK4 (3 µM), menadione (3 µM) and Lip1 (1 µM) were added 3 h prior to the addition of RSL3. Three hours after the addition of RSL3, cells were incubated with 1.5 μM of BODIPY 581/591 C11 (Thermo Fisher) for 30 min at 37 °C. Subsequently, cells were trypsinized, resuspended in 300 μl of Hanks’ balanced salt solution (HBSS, Gibco), strained through a 40 μm cell strainer (Falcon tube with cell strainer CAP), and then analysed using a flow cytometer (CytoFLEX and CytExpert 2.4, Beckman Coulter) with a 488-nm laser paired with a 530/30 nm bandpass filter. Data were analysed using FlowJo Software 10 (Treestar).
Cellular lipid hydroperoxides were detected using the fluorescent probe Liperfluo (Dojindo). H9C2 cells were plated onto black, clear-bottom μClear 96-well culture plates (Greiner). After removal of the medium, the cells were incubated in HBSS containing 2 μM of Liperfluo. Subsequently, the cells were incubated with 100 μM BSO and the indicated compounds for 40 h. After the incubation, the cells were washed with HBSS and observed using a BZ-X800 fluorescence microscope (Keyence). The signal intensity per cell was measured with ImageJ software v1.53 (NIH).
Iron-chelating activity assay
Iron-chelating activity was measured by Metalloassay kit Fe (FE02M, Metallogenics). After the addition of the compounds (final concentration, 100 μM) into an iron standard solution of 200 μg dl−1 of iron(iii) nitrate, free iron levels in the solution were measured according to the protocol.
Lipids from cells were extracted using the methyl-tert-butyl ether (MTBE) method37. In brief, cell pellets (4 to 6 × 106 cells) collected in phosphate-buffered saline (PBS) containing dibutylhydroxytoluene (BHT, 100 µM) and diethylenetriamine pentaacetate (100 µM) were washed and centrifuged. Splash Lipidomix (Avanti Polar Lipids) was added (5 μl) and incubated on ice for 15 min. After ice-cold methanol (375 µl) and MTBE (1,250 µl) were added, samples were vortexed and incubated for 1 h at 4 °C (Orbital shaker, 32 rpm). Phase separation was induced by the addition of water (375 μl), vortexed, incubated for 10 min at 4 °C (Orbital shaker, 32 rpm), and centrifuged to separate organic and aqueous phase (10 min, 4 °C, 1,000g). The organic phase was collected, dried in the vacuum concentrator and redissolved in 53 μl of isopropanol, centrifuged and 50 μl were transferred in glass vials for LC–MS analysis.
Lipids from mouse livers (approximately 150 mg wet tissue weight) were extracted according to Folch extraction method38. SPLASH LIPIDOMIX (Avanti Polar Lipids, 30 μl) was added. Samples were homogenized in methanol (1 ml) by cryomilling and transferred in 10 ml glass tubes. Lysis beads were washed with methanol (400 μl) and chloroform (1,000 μl). Additional 1.8 ml chloroform was added, samples were vortexed (2 min, 2500 rpm) and incubated for 1 h at 4 °C with rotation (32 rpm). Phase separation was induced by adding water (840 μl). Samples were mixed by vortexing and incubated for 10 min 4 °C with rotation, before centrifugation (10 min, 1,000g, 4 °C). The lower, organic phase containing lipids was collected into new glass vials. For re-extraction, chloroform (2.8 ml) was added, samples vortexed, incubated (1 h, 4 °C, and 32 rpm), and centrifuged (10 min, 1,000g, 4 °C). The organic phases, combined from both extractions, were dried in a vacuum. Lipids were reconstituted in 300 μl IPA, and transferred in glass vials for LC–MS analysis. To avoid oxidation, all solvents used for lipid extraction were spiked with 0.1% (w/v) BHT and cooled on ice before use.
Reversed phase liquid chromatography (RPLC) was carried out on a Vanquish focused+ (Thermo Fisher Scientific) equipped with an Accucore C30 column (150 × 2.1 mm; 2.6 µm, 150 Å, Thermo Fisher Scientific). Lipids were separated by gradient elution with solvent A (acetonitrile/water, 1:1, v/v) and B (isopropanol/acetonitrile/water, 85:15:5, v/v) both containing 5 mM NH4HCO2 and 0.1% (v/v) formic acid. Separation was performed at 50 °C with a flow rate of 0.3 ml min−1 using the following gradient: 0–20 min, 10 to 86% B (curve 4); 20–22 min, 86 to 95% B (curve 5); 22–26 min, 95% isocratic; 26–26.1 min, 95 to 10% B (curve 5); followed by 5 min re-equilibration at 10% B. Mass spectrometry analysis was performed on Thermo Scientific Q Exactive Plus Quadrupole-Orbitrap (Thermo Fisher Scientific) equipped with a heated electrospray (HESI) source and operated in negative ion mode with the following parameters: sheath gas 40 arbitrary units, auxiliary gas 10 arbitrary units, sweep gas 1 arbitrary units, spray voltage 2.5 kV, capillary temperature 300 °C, S-lens RF level 35%, and aux gas heater temperature 370 °C. For relative quantification of oxidized lipids, retention time scheduled parallel reaction monitoring using elemental composition of previously identified or computationally predicted oxidized lipids as precursors was used in negative ion mode at the resolution of 17,500 at m/z 200, AGC target of 2 × 105 and a maximum injection time of 200 ms. The isolation window for precursor selection was 1.2 m/z, and normalized stepped collision energy (20–30–40 and 30–40–50 eV for phospholipids and neutral lipids, respectively) was used for HCD. Data were acquired in profile mode.
Acquired data were processed by Skyline v. 21.139 considering fragment anions of oxidized fatty acyl chains as quantifiers. The obtained peak areas were normalized by appropriate lipid species from SPLASH LIPIDOMIX Mass Spec Standard (Avanti), e.g. by LPC(18:1(d7)), LPE(18:1(d7)), PC(15:0/18:1(d7)), or phosphatidylethanolamine (15:0/18:1(d7)), and the sample weights. Normalized peak areas were further log-transformed and auto-scaled in MetaboAnalyst online platform v5.0 (https://www.metaboanalyst.ca)40. Zero values were replaced by 0.2× the minimum values detected for a given oxidized lipid within the samples. Oxidized lipids showing a significant difference (ANOVA, adjusted P-value (false discovery rate (FDR)) cutoff: 0.05) between samples were used for the heat maps. The heat maps were created in Genesis v1.8.1 (Bioinformatics TU-Graz)41. The colour scheme corresponds to auto-scaled log fold change relative to the mean log value within the samples. Shorthand notations for oxidized lipids are given using LipidLynxX system v0.9.2442.
Cells were lysed in LCW lysis buffer (0.5% Triton X-100, 0.5% sodium deoxycholate salt, 150 mM NaCl, 20 mM Tris-HCl, 10 mM EDTA, 30 mM sodium pyrophosphate tetrabasic decahydrate) containing protease and phosphatase inhibitor mixture (cOmplete and phoSTOP, Roche), and centrifuged at 15,000g, 4 °C for 20 min. The supernatant was collected and used as the protein sample. Western blotting was performed by standard immunoblotting procedure with 12% SDS–PAGE gel, PVDF membrane, and primary antibodies against GPX4 (1:1,000, ab125066, Abcam), 4HNE (1 µg ml−1, MHN-20P, JaICA), human FSP1 (1:1,000, sc-377120, Santa Cruz), mouse FSP1 (1:100, clone 1A1 rat IgG2a; and 1:2, hybridoma supernatant of clone 14D7 rat IgG2bƙ, developed in-house), VKORC1 (1:1,000, ab206656, Abcam), GGCX (1:1,000, ab197982, Abcam), β-actin–HRP (1:5,000, A3854, Sigma-Aldrich) and valosin containing protein (VCP, 1:10,000, ab11433, Abcam). Images were analysed with Image Lab 6.0 software (Bio-Rad).
Generation of monoclonal antibodies against mouse Fsp1
Female Wistar rats (RjHan:Wi, age 160 days) were immunized subcutaneously and intraperitoneally with a mixture of 70 µg recombinant C-terminal-His tagged full-length mouse Fsp1 protein in 200 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL), and 200 μl Incomplete Freund’s adjuvant (Sigma-Aldrich). After 8 weeks, a boost without Freund’s adjuvant was given intraperitoneally and subcutaneously 3 days before fusion. Fusion of the myeloma cell line P3X63-Ag8.653 (CRL-1580, ATCC) with the rat immune spleen cells was performed using polyethylene glycol 1500. After fusion, the cells were plated in 96-well plates using RPMI 1640 medium with 20% FBS, glutamine, pyruvate, non-essential amino acids and HAT media supplement (Hybri-Max, Sigma-Aldrich). Hybridoma supernatants were screened 10 days later in a flow cytometry assay for binding to c-His tagged Fsp1 protein captured via biotinylated mouse anti-His antibody (clone HIS 3D5, prepared in-house) to streptavidin beads (PolyAN). Hybridoma supernatant was incubated for 90 min with beads and Atto-488-coupled subclass-specific monoclonal mouse-anti-rat IgG. Antibody binding was analysed using ForeCyt software 8 (Sartorius). Positive supernatants were further validated by Western blotting. Selected hybridoma cells were subcloned by limiting dilution to obtain stable monoclonal cell lines.
Production of purified recombinant human FSP1
Recombinant human FSP1 protein (rhFSP1) was produced in Escherichia coli, and purified by affinity chromatography with a Ni-NTA system as described previously4.
Liposomes were prepared from egg phosphatidylcholine (egg PC, Sigma-Aldrich) in pH 7.4 TBS buffer (25 mM, extruded to 100 nm, Chelex-100 treated) according to our previous report25,43. Liposomes (from the above suspension), STY-BODIPY (from a 1.74 mM stock in DMSO) and the test quinone (from appropriate stock solutions in CH3CN) were combined and diluted to 285 μl with pH 7.4 TBS buffer in the wells of a 96-well plate, such that the concentrations in the well were 1.0526 mM liposomes, 1.0526 μM STY-BODIPY and 2.1053, 4.2105, 8.4210 or 16.8421 μM quinone. This was followed by the addition of 5 μl rhFSP1 at desired concentrations (with 19.2 μM FAD in pH 7.4 TBS buffer). The plate was incubated at 37 °C in a plate reader for 1 min followed by a vigorous mixing protocol for 5 min. The plate was ejected from the plate reader, and 5 μl of NADH (appropriate concentrations in pH 7.4 TBS buffer) and 5 μl of DTUN (12 mM in ethanol) were added such that the final concentrations of reagents were: 1 mM liposomes, 1 μM STY-BODIPY, 2, 4, 8 or 16 μM quinone, 2, 4, 8, 16 or 32 nM rhFSP1, 320 nM FAD, 4, 8, 16, 32 or 64 μM NADH and 200 μM DTUN. The plate was incubated at 37 °C for 1 min followed by another 1 min wherein it was mixed and the fluorescence (λex/λem = 488/518 nm) recorded every 2 min for the duration of the experiment. For determinations of inhibition rate constants, the rate of initiation (Ri) under the exact experimental conditions was first determined from the inhibition period observed upon inclusion of PMC, for which n = 2, as a representative data trace is shown in Extended Data Fig. 7a. The Ri was calculated from the expression below to yield Ri = 7.81 × 10−10 s−1 from tinh = 10,240 s, where tinh is the inhibited period. This Ri was used along with the expression in Extended Data Fig. 7a to calculate the rate constants shown in Extended Data Fig. 7i .
Synthesis of 1,4-dimethoxy-2-methylnaphthalene
To a solution of 2-bromo-1,4-dimethoxy-3-methylnaphthalene44 (dimethylmenadione) (600 mg, 2.13 mmol) in THF (20 ml) was added n-butyllithium (1.02 ml, 2.5 M in n-hexane) dropwise at −78 °C; the mixture was stirred at −78 °C for 10 min, followed by the addition of 0.5 ml water. The cooling bath was removed and reaction mixture was warmed to room temperature. Solvent was removed, and the mixture was purified by column with ethyl acetate/hexanes as the eluent. 1,4-Dimethoxy-2-methylnaphthalene was obtained as a light-yellow oil (350 mg, 81% yield). 1H NMR (400 MHz, chloroform-d) δ 8.20 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.54–7.49 (m, 1H), 7.45–7.41 (m, 1H), 6.61 (s, 1H), 3.97 (s, 3H), 3.87 (s, 3H), 2.45 (s, 3H). 13C NMR (101 MHz, chloroform-d) δ 151.7, 147.1, 128.8, 126.6, 125.7, 125.4, 124.7, 122.3, 121.6, 106.9, 61.4, 55.7, 16.4.
Synthesis of vitamin K–coumarin conjugates
2-(Hydroxymethyl)-3-methylnaphthalene-1,4-dione44 (42 mg, 0.20 mmol) and 7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl chloride4 (67 mg, 0.24 mmol) were dissolved in CH2Cl2 (2 ml), followed by the addition of Et3N (40 μl, 0.003 mol) and N,N-dimethylpyridin-4-amine (3 mg, 0.024 mmol). The mixture was stirred at room temperature for 12 h. Precipitation was formed during this process. The reaction mixture was filtered and washed with ether. Crude product was purified by recrystallization from ethyl acetate to give vitamin K–coumarin as a yellow solid (55.2 mg, 62% yield). 1H NMR (600 MHz, chloroform-d) δ 8.38 (s, 1H), 8.12–8.10 (m, 2H), 7.75–7.72 (m, 2H), 7.33 (d, J = 9.1 Hz, 1H), 6.64 (dd, J = 9.1, 2.4 Hz, 1H), 6.47 (d, J = 2.4 Hz, 1H), 5.40 (s, 2H), 3.44 (q, J = 7.1 Hz, 4H), 2.37 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz, Chloroform-d) δ 185.3, 183.6, 163.7, 158.6, 158.1, 152.8, 149.5, 148.1, 139.6, 133.9, 133.8, 132.3, 132.0, 131.4, 126.7, 126.6, 110.2, 108.4, 108.2, 97.4, 58.2, 45.6, 13.2, 12.5. HRMS–ESI (m/z) calculated for C26H23NNaO6 [M+Na]+: 468.1423, found 468.1430.
Synthesis of CoQ–coumarin conjugates
2-(Hydroxymethyl)-5,6-dimethoxy-3-methylcyclohexa-2,5-diene-1,4-dione45 (43 mg, 0.20 mmol) and 7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl chloride46 (67 mg, 0.24 mmol) were dissolved in CH2Cl2 (2 ml), followed by the addition of Et3N (40 μl, 0.003 mol) and N,N-dimethylpyridin-4-amine (3 mg, 0.024 mmol). The mixture was stirred at room temperature for 12 h. Precipitation was formed during this process. The reaction mixture was filtered and washed with ether. Crude product was purified by recrystallization from ethyl acetate to give CoQ–coumarin as a yellow solid (49.1 mg, 54% yield). 1H NMR (600 MHz, Chloroform-d) δ 8.36 (s, 1H), 7.34 (d, J = 8.9 Hz, 1H), 6.61 (dd, J = 9.0, 2.4 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 5.20 (s, 2H), 4.03 (s, 3H), 3.99 (s, 3H), 3.44 (q, J = 7.1 Hz, 4H), 2.19 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz, Chloroform-d) δ 184.3, 183.0, 163.6, 158.7, 158.1, 153.1, 149.6, 144.8, 144.8, 143.9, 135.4, 131.4, 109.9, 108.0, 107.9, 97.0, 61.4, 61.4, 57.6, 45.4, 12.5, 12.5. HRMS–ESI (m/z) calculated for C24H25NNaO8 [M+Na]+: 478.1478, found 478.1480.
Monitoring FSP1 activity with quinone–coumarin conjugates
FAD, NADH and rhFSP1 in pH 7.4 TBS buffer were added in succession to varying concentrations of vitamin K–coumarin or CoQ–coumarin conjugate in pH 7.4 TBS buffer at 37 °C (final concentration: 6 nM rhFSP1, 50 nM FAD, 200 μM NADH) and the initial rates of the reaction were obtained by monitoring the increase in the fluorescence upon the reduction of the quinone to the hydroquinone on a plate reader (λex/λem = 415/470 nm). The raw fluorescence data were converted to hydroquinone concentrations using response factors of 4.64 × 109 RFU μM−1 (for CoQ–coumarin) and 3.40 × 109 RFU μM−1 (for vitamin K–coumarin) which were determined from a standard curve obtained from the maximum fluorescence recorded for various concentrations of the quinones in the presence of massive excess of either rhFSP1, FAD or NADH.
LipiRADICAL Green assay
LipiRADICAL Green assay was performed according to a previous report27 using the fluorescence probe LipiRADICAL Green, previously called NBD-Pen (FDV-0042, Funakoshi) with several modifications. Arachidonic acid (10931, Sigma-Aldrich) and soybean lipoxygenase (LOX) from Glycine max (L7395, Sigma-Aldrich) were used in an AA/LOX system. Ninety microlitres of PBS pH 7.4 containing 5 μM LipiRADICAL Green, 100 μg ml−1 LOX, and the indicated final concentration of compounds were prepared in black-walled 384 well plates. Immediately after the addition of a 10 μl solution of 5 mM AA (final 500 μM) to the mixture, the fluorescence intensity (ex 470/em 530 nm) was measured every 30 s at 37 °C using a Spectra Max M5 plate reader (Molecular Devices). The intensity before the addition of AA was used as background. For the phosphatidylcholine hydroperoxide (PCOOH)/Fe2+ system, PCOOH was enzymatically synthesized from 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, Avanti Polar Lipids) using soybean lipoxygenase-1 and chromatographically purified47. Ninety microlitres of water containing 5 μM LipiRADICAL Green, 10 μM PCOOH and the indicated final concentration of compounds were prepared in black-walled 384 well plates. Immediately after the addition of 10 μl solution of 500 μM of Fe(NH4)2(SO4)2 (final 50 μM) to the mixture, the fluorescence intensity was measured as described above. For LipiRADICAL Green assay using rhFSP1, NADH and AA/LOX system, 80 μl of PBS containing 5 μM LipiRADICAL Green, 200 μM NADH, 150 nM hFSP1, and 100 μg ml−1 LOX were prepared. After the addition of 10 μl solution of 1 mM phylloquinone or MK4 (final 100 μM) and 10 μl solution of 5 mM AA (final 500 μM) to the mixture, the kinetics of LipiRADICAL Green fluorescence intensity was measured. For the PCOOH/Fe2+ system, 80 μl of PBS containing 5 μM LipiRADICAL Green, 200 μM NADH, 150 nM hFSP1, and 20 μM PCOOH were prepared. After the addition of 10 μl solution of 3 mM phylloquinone or MK4 (final 300 μM) and 10 μl solution of 500 μM of Fe(NH4)2(SO4)2 (final 50 μM) to the mixture, the fluorescence intensity was measured.
FSP1 activity assay by measuring NADH consumption
FSP1 enzymatic assay was performed as described with a minor modification4. NADH consumption was measured at 340 nm using 100 μl of enzyme reactions in PBS pH 7.4 on a 96-well plate. Enzyme reactions contained 150 nM rhFSP1, 200 μM NADH (freshly prepared in water) and 300 μM of different substrate candidates (phylloquinone, MK4, and menadione). A Spectra Max M5 Microplate Reader (Molecular devices) was used to determine the absorbance at 340 nm every 30 s. Reactions without NADH or without enzyme were used to normalize the results.
FSP1 activity inhibitor assay
Enzyme reactions in PBS containing 150 nM rhFSP1, 200 μM NADH and the inhibitors (iFSP1, warfarin and dicoumarol) were prepared. After the addition of 100 μM resazurin sodium salt (Sigma-Aldrich), fluorescent intensity (ex 540/em 590 nm) was measured every 30 s.
Chemical reduction of menadione
Menadione and menadiol (300 μM) were incubated with 1 mM DTT or 10 mM GSH in water at room temperature for 5 min, and then measured by absorbance spectrum ranging from 200 to 450 nm by using a Spectra Max M2e (Molecular Devices). Background control (in blank well) of absorbance values was subtracted from each individual absorbance value.
Detection of MK4-H2 by FIA–MS
To detect chemical reduction of MK4, 22.5 mM of MK4 (dissolved in chloroform, 10 μl) was diluted in methanol (190 μl), then 1 mg NaBH4 was added. Reactant solution (10 μl) was collected before and 1, 15, 30, 60, and 120 min after the addition of NaBH4, and dissolved in methanol (990 μl). The sample solution was analysed by flow injection analysis mass spectrometry (FIA-MS) using a LC–MS/MS system consisted of an Exion LC system connected to a QTRAP 6500+ tandem mass spectrometer (SCIEX). To detect enzymatic reduction of MK4, 1 mM MK4 (dissolved in DMSO, 10 μl) was added with 170 μl PBS, 1.5 μM rhFSP1 (dissolved in PBS, 10 μl), and 2 mM NADH (dissolved in DDW, 10 μl). The solution was incubated at 37 °C for 30 min. After incubation, a part of the reactant solution (10 μl) was dissolved in methanol (990 μl) and analysed by FIA-MS. Mass spec parameters are described in Supplementary Table 2.
Quantification of cellular MK4 and MK4 epoxide levels
HepG2 cells (1 × 106 cells per well) were seeded on 6 well plates. On the next day, medium was replaced with fresh medium with or without warfarin (10 µM). On the following day, cells were incubated in the presence or absence of MK4 (3 µM) for 7 h. After washing with PBS three times, cells were trypsinized and collected. Cell pellets were suspended in 400 µl PBS, supplemented with 20 µl of MK4-d7 (2 ng µl−1 in ethanol, 25709, Cayman) as internal standard, and sonicated for 30 s with a sonication probe (Bronson Sonifer). In this procedure, 10 µl of cell lysate was analysed for protein determination with a BCA protein assay (Pierce BCA Protein Assay Kit, Thermo Fisher). Extraction of vitamin K and its metabolites from cells was performed as reported33. Four-hundred microlitres ethanol and 1.2 ml hexane were added to the cell lysate (in PBS, 400 µl) followed by shaking for 5 min. Samples were centrifuged at 1,000g for 5 min, and the upper organic layer was collected. Re-extraction of the remaining aqueous phase was performed by addition of 150 µl ethanol and 450 µl hexane with subsequent vortexing. Samples were centrifuged at 1,000g for 5 min. Collected organic layers were combined, spiked with 20 µl of phylloquinone (2 ng µl−1 in ethanol) as recovery standard and evaporated under reduced pressure. Dried extracts were resuspended in 30 μl ethanol. Quantification of the target analytes (MK4 and MK4 epoxide) was achieved using an Agilent 5890 Series II gas chromatograph (GC) coupled with a Thermo Finnigan SSQ7000 single quadrupole mass spectrometer (MS). Chromatographic separation was carried out on a Restek Rtx-5Sil MS column (30 m × 0.25 mm internal diameter × 0.25 µm film thickness). Two microlitres of each sample was injected in splitless mode using helium as carrier gas at a constant pressure of 16 psi. The injection temperature was 280 °C. Initial column temperature was 90 °C held for 1.5 min, increased to 220 °C at a rate of 20 °C min−1, followed by a second ramp to 320 °C at a rate of 10 °C min−1 and held for 10 min. The mass spectrometer was operated in negative chemical ionization mode and the masses of the negative molecular ions were registered in single ion monitoring mode.
Quantification of MK4 and MK4 epoxide in mouse samples
Blood samples of mice were collected by bleeding from the retroorbital plexus into citrate-treated tubes. After centrifugation (3,000g for 10 min), plasma samples were obtained and stored −80 °C until analysis. Liver tissues were collected from mice after transcardiac perfusion with 10 ml PBS, snap-frozen into liquid nitrogen and stored at −80 °C. For sample preparation of plasma, 100 µl aliquots of plasma were transferred into glass tubes, spiked with 20 ng of MK4-d7 and briefly mixed. Next, 2 ml ethanol, 4 ml hexane and 100 µl water containing butylated hydroxytoluene (0.1 %, w/v) were added. After vigorously mixing for 5 min, the samples were centrifuged at 2,200g for 5 min at 4 °C. The upper layer was transferred into a clean glass tube, and the samples were then re-extracted by the addition of an equal volume of hexane. Both supernatants were collected and evaporated in vacuo. The samples were dissolved in 2 ml hexane and loaded onto silica columns. For sample preparation of tissues, the tissues (kidney and liver) were weighed, transferred to lysing matrix tubes containing stainless steel beads (MP Biomedicals), and then thoroughly homogenized in 1 ml ethanol containing 20 ng of MK4-d7. The tissue homogenates were transferred into glass tubes using glass Pasteur pipettes. Following the addition of 6 ml acetone containing BHT (0.1 %, w/v), the homogenates were thoroughly mixed using a Ohaus Multi-Tube Vortex mixer, for 5 min at 2,500 rpm, allowed to stand for 5 min, and centrifuged at 2,200g for 5 min at 4 °C. This procedure was repeated three times. Supernatants were collected and evaporated in vacuo. The samples were dissolved in 2 ml water and 6 ml hexane containing BHT (0.1 %, w/v), thoroughly mixed, and centrifuged at 2,200g for 5 min at 4 °C. The samples were evaporated in vacuo, dissolved in 2 ml hexane and loaded onto silica columns. Plasma and tissue extracted samples were applied to silica Sep-Pak extraction cartridges (500 mg per 3 ml, Waters) connected to a Visiprep SPE Vacuum Manifold (Supelco), which were preconditioned prior with 3 ml diethyl ether:hexane (1:1, v/v) and then 3 × 2 ml hexane. After sample application, the cartridges were washed with 2 ml hexane followed by 4 × 2 ml hexane containing BHT (0.1 %, w/v) to remove concomitants. The vitamin K-containing fraction was then eluted with 4 ml diethyl ether:hexane (3:97, v/v). The eluate was evaporated in vacuo and the residue reconstituted in 100 µl water:methanol (2:98, v/v) for measurement with LC–MS/MS. As vitamin K is light sensitive, samples were protected from light during preparation and analysis. MK4 and MK4 epoxide were separated by reversed phase liquid chromatography (RPLC) on a Sciex Exion LC System equipped with a Kinetex F5 100 × 2.1 mm, 100 Å, 2.6 µm column (Phenomenex). Analytes were separated by gradient elution with mobile phase A (H2O containing 5 mM ammonium formate) and B (methanol), both containing 0.1% (v/v) formic acid. Separation was performed at 50 °C with a flow rate of 0.5 ml min−1 using the following gradient: 0–1 min, 70 to 98% B; 1–3 min, 98% isocratic; 3–3.1 min, 98 to 70% B; and 3.1–5 min, 70% isocratic. MK4 and MK4 epoxide were quantified by LC–MS/MS electrospray ionization on a Sciex Triple Quad 7500 LC–MS/MS System, operating in positive mode. Settings were as follows: CUR 50 psi, IS 3,500 V, TEM 500 °C, GS1 20 psi, GS2 70 psi, MRM dwell time 55 ms, pause between mass range 5 ms and EP 10 V. The following parent-to-daughter transitions were monitored: m/z 452.4 [M+H]+ to m/z 194.0 for MK4-d7 with CE of 34 V and CXP of 10 V, m/z 445.1 [M+H]+ to m/z 187.0 for MK4 with CE of 31 V and CXP of 10 V, m/z 461.2 [M+H]+ to m/z 161.0 for MK4 epoxide with CE of 34 V and CXP of 14 V. The limits of quantification for MK4 were 0.2 ng mg−1 tissue and 0.1 ng ml−1 plasma, and those for MK4 epoxide were 2.0 ng mg−1 tissue and 1.0 ng ml−1 plasma.
All experiments were performed in compliance with the German Animal Welfare Law and have been approved by the institutional committee on animal experimentation and the government of Upper Bavaria (approved no. ROB‐55.2‐2532‐Vet_02‐18‐13 and ROB-55.2-2532.Vet_03-17-68) and the State of Bavaria (permission granted by the government of Lower Franconia, approved No. 54-2532.1-19/13), the Landesdirektion Sachsen (TVV07/2021) involving an independent ethics committee and the Animal Committee of Tohoku University (approved No. 2019-BeA012, 2019-BeA014 and 2019PhA-010-01). Mice were kept under standard conditions with water and food ad libitum and in a controlled environment (22 ± 2 °C, 55 ± 5% humidity, 12 h light/dark cycle). For animal studies, mice were randomized into separate cages. Sex-matched littermates were used and experiments were intended to test a single variable.
Hepatocyte-specific inducible Gpx4-KO mice
To generate mice with a TAM-inducible hepatocyte-specific deletion of Gpx4 (Alb-creERT2;Gpx4fl/fl), Gpx4fl/fl mice were first crossbred with Alb-creERT2 mice48 (kindly provided by P. Chambon) to yield Alb-creERT2;Gpx4fl/+ mice. These were then crossed with Gpx4fl/fl mice to generate Alb-creERT2;Gpx4fl/fl mice and respective controls. To achieve inducible disruption of the floxed Gpx4 alleles, mice were intraperitoneally injected with 2 mg TAM (dissolved in Miglyol 812, Caelo) on two consecutive days. Animals were equally distributed between sex and weight and were typically 8–10 weeks of age. For pharmacological treatment, vehicle or MK4 (100 mg kg day−1 dissolved in Miglyol, twice daily) was intraperitoneally administrated to the mice each day starting from 2 days before the first TAM injection until the completion of the study. The diet was changed from a standard diet (containing 143 mg kg−1 vitamin E, no. 1314 Fortified, Altromin) to a vitamin E-deficient diet (containing <7 mg kg−1 vitamin E, E15314-247, ssniff Spezialdiäten) at the timing of the first TAM injection. When animals reached the humane end point, they were immediately euthanized. For the end point analysis, the mice were euthanized 7 days after the first TAM injection, and the plasma and tissues were collected. Serum ALT were measured by AU480 chemistry analyser (Beckman Coulter). For the pharmacokinetic study of MK4, samples of plasma, liver and kidney were collected from Gpx4fl/fl mice 0, 1, 3, 6 and 24 h after an intraperitoneal injection of MK4 (200 mg kg−1 dissolved in Miglyol).
Liver ischaemia–reperfusion injury model in mice
Eight to 10-week-old male C57BL/6J mice, provided by Charles River (Germany), were fed a standard diet (containing 135 mg kg−1 vitamin E, ssniff Spezialdiäten) and underwent liver ischaemia–reperfusion injury as described previously10. In brief, mice were aneasthetized with xylazine/ketamine and shaved at their front. After opening the abdominal cavity an atraumatic clip was placed across the portal vein, hepatic artery and bile duct, just above branching to the right lateral lobe. After 90 min of ischaemia, the clamp was removed and the liver was reperfused. Mice were euthanized 24 h following transient ischaemia–reperfusion and blood and tissues were collected. MK4 (200 mg kg−1 dissolved in Miglyol 812) or vehicle was injected intraperitoneally 24 h and 1 h before the onset of ischaemia. Serum ALT was measured using a Dimension 1500 Vista Analyzer (Siemens). Calculation of the necrotic/damaged areas (% of the whole section minus the major vessels) in the haematoxylin and eosin-stained sections were performed in a blinded manner using ZEISS Axio Vision software AxioVs v4.9 (Carl Zeiss).
Kidney ischaemia–reperfusion injury model in mice
Eight- to twelve-week-old male C57BL/6N mice (Charles River), were fed a standard diet (containing 135 mg kg−1 vitamin E, V1534-300, ssniff Spezialdiäten) and underwent renal ischaemia–reperfusion injury as described previously49. In brief, bilateral renal pedicle clamping was performed via a midline abdominal incision for 36 min. Throughout the surgical procedure, the body temperature was maintained between 36 and 37 °C. After removal of the clamps, the abdomen was closed allowing restoration of blood flow as also visually observed. Sham-operated mice underwent the identical surgical procedures, except clamping of renal pedicles. All mice were killed 48 h after the reperfusion. All ischaemia–reperfusion experiments were performed in a double-blinded manner. MK4 (200 mg kg−1 dissolved in corn oil) or vehicle was injected intraperitoneally 1 h before the onset of ischaemia. Serum creatinine and urea were measured in the Institute for clinical chemistry of the University Hospital Dresden (Germany). Kidney tissue damage was quantified by two researchers in a double-blind manner on a scale ranging from 0 (unaffected tissue) to 10 (severe organ damage). The following parameters were chosen as indicative of morphological damage to the kidney after ischaemia–reperfusion injury: brush border loss, red blood cell extravasation, tubule dilatation, tubule degeneration, tubule necrosis, and tubular cast formation. These parameters were evaluated on a scale of 0–10, which ranged from not present (0), mild (1–4), moderate (5 or 6), severe (7 or 8), to very severe (9 or 10). For the scoring system, tissues were stained with periodic acid–Schiff (PAS), and the degree of morphological involvement in renal failure was determined using light microscopy.
Generation of Fsp1
Fsp1−/− mice (that is, B6.129-Aifm2tm1Marc/Ieg) were obtained from Infrafrontier (https://www.infrafrontier.eu; EM:05283). In these mice, exons 5 and 6 of the Aifm2 (also known as Fsp1) gene were replaced by a lacZ-neo cassette. For genotyping PCR, following primers were used: 5′-GCCTGGTATTCACATTGGAA and 5′-GAGTGGATAAGAGTGACCTG for the wild-type allele; 5′-CCGCTTAAGCTAGCCATGGGTAATTC and 5′-GACAGTATCGGCCTCAGGAA for the KO allele.
Warfarin treatment of mice
Sex- and age-matched littermates (20–30 g, aged 8–16 weeks) of Fsp1−/− and Fsp1+/− mice were used. Mice were orally administered with warfarin sodium through bottled drinking water (0.33 mg ml−1 water) until completion of the study. This dose corresponds to a warfarin uptake of 50 mg kg−1 per mouse for a 24-h feeding period, assuming water consumption is 15 ml per 100 g per 24 h. Prothrombin time was measured using CoaguChek Pro II (Roche Diagnostics), which has a reportable range of 9.6 to 96 s, at the timing of 60 h after the start of warfarin sodium administration and 12 h after the subcutaneous injection of 20 mg kg−1 MK4 (dissolved in Miglyol 812) or vehicle. When the prothrombin time was above the detectable limit, the value was regarded as 96 s for the statistical analysis. For the measurement of MK4 and MK4 epoxide levels, plasma and liver tissues were collected 6 h after injection of MK4 (20 mg kg−1, subcutaneous injection) from Fsp1+/− and Fsp1−/− mice treated with warfarin sodium. For the survival study, MK4 (10 mg kg day−1, subcutaneous injection) or vehicle was administrated each day. All mice were monitored twice daily for survival. When animals reached the humane end point, they were immediately euthanized.
Histology, immunohistochemistry and TUNEL staining
Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. For immunohistochemistry, deparaffinized sections were immunolabeled using antibodies for anti-GPX4 (ab125066, Abcam), anti-4HNE (HNEJ-2, JaICA), anti-KIM-1 (AF1817, R&D) and anti-cleaved caspase-3 (9661, Cell Signaling). For anti-GPX4, KIM-1 and cleaved caspase-3 staining, the sections were heated for antigen retrieval in a microwave oven in 0.01 M citrate buffer pH 6.0 (for anti-GPX4 and KIM-1) or in Target Retrieval Solution (S1699, DAKO; for anti-cleaved caspase-3) for 20 min. After blocking with 5% FBS (for GPX4, KIM-1 and cleaved caspase-3) or 10% FBS (for 4HNE) in Tris-buffered saline, pH 7.4 containing 0.01% Tween-20 for 30 min, the sections were incubated with the primary antibodies (anti-GPX4 1:100; anti-4HNE 0.5 µg ml−1; anti-KIM-1 1:200; and anti-cleaved caspase-3 1:100) overnight at 4 °C. After incubation with 0.3% H2O2 in methanol for 20 min, the sections were incubated with the following secondary antibodies for 30 min: biotinylated goat anti-rabbit IgG (1:250; BA-1000, Vector Laboratories) for anti-GPX4; biotinylated goat anti-mouse-IgG (1:200; BA-9200, Vector Laboratories) for anti-4HNE; and biotinylated donkey anti-goat-IgG diluted (1:500; 208000, Abcam) for anti-KIM-1, and then incubated with streptavidin–biotin peroxidase complex (VECTASTAIN Elite ABC system, Vector Laboratories). For anti-cleaved caspase-3 staining, Histofine Simple Stain MAX PO (R) Anti-Rabbit (Nichirei) was used as secondary antibody. The sections were visualized with nickel-enhanced 3,3′-diaminobenzidine (DAB, SK-4100, Vector Laboratories) for anti-GPX4 and KIM-1, or DAB and counterstaining with Mayer’s Hematoxylin for anti-4HNE and cleaved caspase-3. TUNEL staining was performed using the ApopTag peroxidase in situ apoptosis detection kit (Millipore). To reduce false-positive signals, the TdT enzyme was diluted 1:16 in reaction buffer for preparation of the working solution. Gr-1+ cells were immunohistochemically stained on acetone-fixed frozen liver sections. Dried sections were blocked with 10% goat serum for 1 h, and then incubated with anti-Gr-1-FITC antibody (0.5 mg ml−1, 553127, BD Pharmingen) for 30 min at room temperature. The sections were treated with goat anti-Rat Alexa Fluor 488 IgG (H+L) (1:500, A-11006, Invitrogen) and DAPI (5 mg ml−1) for visualization. Gr-1+ cells were counted per high-power field (HPF) (2,000× magnification; five HPF per slide). A blinded scientist received the slides randomly and performed all cell counting procedures.
Quantification and statistical analysis
Statistical information for individual experiments can be found in the corresponding figure legends. Values are presented as mean ± s.d. Statistical comparisons between groups were analysed for significance by two-tailed Student’s t-test, one-way ANOVA with Dunnett’s post hoc test. Survival analysis was done according to the log-rank test. Results were considered significant at P <0.05. Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software) and JMP15 (SAS Institute) software.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.