Journal: Nature medicine

Article Title: Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

doi: 10.1038/nm.3610

Figure Lengend Snippet: Differential regulation of HCV strains by SPHK2-mediated lipid peroxidation. ( a ) Dose-dependent effects of PUFAs on H77S.3/GLuc and HJ3-5/GLuc RNAs in Huh-7.5 cells. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. ( b ) Growth kinetics of H77S.3/GLuc and HJ3-5/GLuc RNAs in the presence of 50 μM PUFAs. Data shown are mean ± s.e.m. of GLuc activity in supernatant fluids of two replicate cultures. ( c ) Cells transfected with HCV RNAs encoding GLuc were treated with DMSO, 100 μM LA or 100 μM LA plus 1 μM SKI. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. L.O.D. = limit of detection. ( d ) Effect of 1 μM SKI, 1 μM VE, 100 μM CoQ10 or 50 μM ARA or DHA, on intracellular malondialdehyde (MDA) abundance in cells transfected with the indicated HCV/GLuc RNAs at 72 h. MDA was significantly increased by PUFAs and reduced by SKI or lipophilic antioxidants ( P < 0.01). ( e ) Analysis of 8-isoprostane abundance in cells electroporated with the indicated HCV RNAs and grown in the presence of 1 μM SKI or VE, or 50 μM LA with or without 1 μM SKI and VE for 48 h. ( f ) Effect of siRNA targeting SPHK isoforms (see ) on MDA accumulation after treatment with increasing concentrations of LA (6.25, 12.5, 25, 50, 100 μM) for 24 h (left panel). MDA levels in Huh-7.5 cells treated with increasing concentrations of LA in the presence of DMSO or 1 μM SKI (right panel). ( g ) Effects of increasing concentrations of VE (left), 1 μM VE alone, or 1 μM VE plus 1 μM SKI (right) on replication of H77S.3/GLuc and HJ3-5/GLuc RNAs. Data shown represent GLuc secreted between 48–72 h relative to DMSO control. ( h ) GLuc secretion from Huh-7.5 cells transfected as in a and treated with 10 μM CuOH with or without 10 μM VE. ( i ) Influence of SKI or VE (each 1 μM) on replication of H77S.3 and HJ3-5 viruses expressing GLuc in cells cultured in the presence or absence of 10% FBS. Medium containing 10% FBS was replaced with FBS-free or 10% FBS media containing SKI, VE or DMSO 6 h after RNA transfection. Data shown represent mean GLuc activity ± s.e.m. from two ( a–f,i ) or three ( g,h ) independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Vitamin E (α-, rac-β-, and γ-tocopherols), 4-deoxypyridoxine hydrochloride (DOP), coenzyme Q10, butylated hydroxytoluene, N-acetyl-L-cysteine, diphenyleneiodonium chloride, oleic acid, and cyclosporine A were from Sigma-Aldrich. nSMase spiroepoxide and cumene hydroperoxide were from Santa Cruz Biotechnology, D609 was from Enzo Life Sciences, and sofosbuvir (PSI-7977) was from Chemscene.

Techniques: Activity Assay, Control, Transfection, Expressing, Cell Culture

Journal: iScience

Article Title: Phospholipase PLA2G7 is complementary to GPX4 in mitigating punicic-acid-induced ferroptosis in prostate cancer cells

doi: 10.1016/j.isci.2024.109774

Figure Lengend Snippet:

Article Snippet: Coenzyme Q10 , Selleck chemicals , Cat# S2398.

Techniques: Virus, Control, Expressing, Plasmid Preparation, Recombinant, Western Blot, Transfection, Saline, Protease Inhibitor, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software, RNA Sequencing

Journal: Toxicology Research

Article Title: Protective effects of coenzyme Q10 and resveratrol on oxidative stress induced by various dioxins on transheterozigot larvae of Drosophila melanogaster

doi: 10.1039/c7tx00027h

Figure Lengend Snippet: Oxidative parameters data obtained from D. melanogaster ( mwh x flr 3 ) for experimental groups with the dioxins and dioxins + antioxidant

Article Snippet: Coenzyme Q10 (CoQ10) and Resveratrol (RSV) were obtained from MP Biomedicals (France).

Techniques:

Journal: Nature Cell Biology

Article Title: FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis

doi: 10.1038/s41556-025-01790-y

Figure Lengend Snippet: a , Thin-layer chromatogram of lipids extracted from LD fractions. b , Parallel reaction monitoring (PRM) used for the relative quantification of known FSP1 substrates in LD fractions generated by density gradient centrifugation from cell lysates. Data are presented as mean ± s.d., n = 3. c , Absolute quantification of CoQ10 levels by PRM in LD fractions spiked with an isotopically labelled CoQ10 internal standard. Levels of CoQ10 were normalized to total amounts of TG and CE lipids, quantified via thin-layer chromatography. Data are presented as mean ± s.d., n = 3. d , Cartoon describing the FENIX assay, adapted for artificial LDs after recruitment of recombinant FSP1. e , FENIX assay in artificial LDs with various substrate and enzyme compositions. f , FENIX assay in artificial LDs in the presence and absence of catalytically active FSP1 or the catalytically inactive FSP1(E156A) mutant. g , FENIX assay in artificial LDs in the presence of 100 nM FSP1, 10 µM CoQ10, 1 mM total lipid and varying concentrations of NADH. NADH consumption throughout the FENIX assay was assessed via NADH autofluorescence within the same experiment. h , i , FENIX assay in artificial LDs in the presence of increasing concentrations of CoQ10 ( h ) or α-tocopherol ( i , αTOC). j , k , PRM-based quantification of TG and PC oxidation products during the peroxidation of artificial LDs. Data are presented as mean ± s.d., n = 3.

Article Snippet: Coenzyme Q10 was extracted as described previously by the addition of 600 μl of methanol (Fisher Scientific, A456) + 0.1% (vol/vol) hydrochloric acid (Fisher Scientific, A144) and 600 μl of hexane (Fisher Scientific, H302).

Techniques: Targeted Proteomics, Quantitative Proteomics, Generated, Gradient Centrifugation, Thin Layer Chromatography, Recombinant, Mutagenesis

Journal: Nature Cell Biology

Article Title: FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis

doi: 10.1038/s41556-025-01790-y

Figure Lengend Snippet: A : Characterization of recombinantly expressed FSP1. Coomassie stained gel indicates high FSP1/FSP1(E156A) purity and western blot analysis proves the identity of FSP1 and the presence of a His-Tag. B : Recombinant FSP1 but not FSP1(E156A) is capable of consuming NADH in the presence of the soluble CoQ10 derivative CoQ1. NADH consumption is assessed by measuring endogenous absorbance of NADH at 340 nm. (data are represented as mean ± SD, n = 3) C : Recombinant FSP1 but not FSP1(E156A) can reduce CoQ to CoQH2 in the presence of NADH. CoQH2 formation is assessed using the fluorescent CoQ-reduction reporter CoQ1-coumarin. (data are represented as mean ± SD, n = 3) D : Recombinant FSP1 can be recruited to nickel-phospholipid doped liposomes. Recruitment was assessed using a liposome flotation assay and gel electrophoresis with silver stain detection. Data shown are representative of two independent replicates. E : FENIX assay is Egg-phosphatidylcholine liposomes in the presence of various substrate and enzyme combinations. (data are represented as mean, n = 3) F : FENIX assay in Egg-phosphatidylcholine liposomes in the presence of catalytically inactive FSP1(E156A). (data are represented as mean, n = 3) G : FENIX assay in Egg-phosphatidylcholine liposomes in the presence of α-tocopherol and/or FSP1. (data are represented as mean, n = 3) H : LipiRadicalGreen assay to detect free radicals generated by the reaction of triglyceride hydroperoxides with iron. LipiRadicalGreen activation was monitored using fluorescence spectroscopy. (data are represented as mean ± SD, n = 3) I : LC-MS/MS chromatogram of the transition of CoQ8 [M + H]+ and [M + NH4]+ adducts to m/z 197.1 for and quantification of CoQ8 and CoQ10 levels in recombinant FSP1 preparations. (data are represented as mean ± SD, n = 3) J : Detection of BSA bound CoQ10 as a model system for copurified coenzyme Q in recombinant FSP1 preparations. CoQ10 was detected via absorbance spectroscopy. BSA/CoQ10 complex was treated with biobeads or 1-butanol (1-BuOH) to delipidate BSA. K : BSA/CoQ10 complex delipidated with 1-butanol. Remaining CoQ10 levels were determined by absorbance at 260 nm. (data are represented as mean ± SD, n = 3) L : Recombinant FSP1 was treated with 1-butanol and protein levels remaining after delipidation were quantified via bichinonic acid assay. (data are represented as mean ± SD, n = 3) M : Measurement of remaining FSP1 activity after delipidation via CoQ1-coumarin assay. (data are represented as mean ± SD, n = 3).

Article Snippet: Coenzyme Q10 was extracted as described previously by the addition of 600 μl of methanol (Fisher Scientific, A456) + 0.1% (vol/vol) hydrochloric acid (Fisher Scientific, A144) and 600 μl of hexane (Fisher Scientific, H302).

Techniques: Staining, Western Blot, Recombinant, Liposomes, Nucleic Acid Electrophoresis, Silver Staining, Generated, Activation Assay, Fluorescence, Spectroscopy, Liquid Chromatography with Mass Spectroscopy, Acid Assay, Activity Assay

Journal: Nature Cell Biology

Article Title: FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis

doi: 10.1038/s41556-025-01790-y

Figure Lengend Snippet: A : Schematic of ethanolic infusion of lipids to synthesize artificial lipid droplets. B : Phase contrast microscopy of artificial lipid droplet preparations. C : Size and polydispersity of artificial lipid droplet preparations assessed by dynamic light scattering. (data are represented as mean ± SD, n = 3) D : Lipid class distribution in lipid droplet fractions extracted from U-2 OS cells treated with 200 µM oleate and 0.1% BSA for 24 h. Lipids were quantified by thin layer chromatography. (data are represented as mean ± SD, n = 3) E : Thin layer chromatograms of lipids artificial lipid droplets. F : Nile red fluorescence in liposomes, artificial lipid droplets and lipid droplets extracted from Huh7 cells. Nile red was excited at 530/10 nm and shows red-shifted fluorescence in comparison to liposomes but similar fluorescence as compared to lipid droplet fractions from cells indicating a lipid droplet like supramolecular structure. G : Schematic describing the strategy to recruit His-tagged FSP1 to nickel-phospholipid spiked artificial lipid droplets. Red phospholipids = DGS-NTA(Ni), red tail on FSP1 = His-Tag. H : Schematic describing artificial lipid droplet flotation to probe successful recruitment of His-tagged FSP1. Lower panel shows silver stained gel detecting FSP1 protein in each collected fraction indicating strong binding. I : FENIX assay in artificial lipid droplets in the presence of 100 nM FSP1, 25 µM NADH and varying concentrations of the alternative FSP1 substrate menaquinone-4 (MK4). (data are represented as mean, n = 3) Data shown are representative of two independent replicates. J : FENIX assay in artificial lipid droplets in the presence of 10 µM CoQ10, 25 µM NADH and varying concentrations of the catalytically dead FSP1 mutant FSP1(E156A). (data are represented as mean, n = 3) K : FENIX assay in artificial lipid droplets in the presence of 10 µM CoQ10, 25 µM NADH and varying concentrations of active FSP1 mutant FSP1. (data are represented as mean, n = 3).

Article Snippet: Coenzyme Q10 was extracted as described previously by the addition of 600 μl of methanol (Fisher Scientific, A456) + 0.1% (vol/vol) hydrochloric acid (Fisher Scientific, A144) and 600 μl of hexane (Fisher Scientific, H302).

Techniques: Microscopy, Thin Layer Chromatography, Fluorescence, Liposomes, Comparison, Staining, Binding Assay, Mutagenesis

Journal: Nature Cell Biology

Article Title: FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis

doi: 10.1038/s41556-025-01790-y

Figure Lengend Snippet: A – I : Quantification of lipid peroxidation products detected by parallel reaction monitoring during peroxidation of artificial lipid droplets in the presence of 25 µM NADH, 10 µM CoQ10 and 100 nM FSP1. (data are represented as mean ± SD, n = 3).

Article Snippet: Coenzyme Q10 was extracted as described previously by the addition of 600 μl of methanol (Fisher Scientific, A456) + 0.1% (vol/vol) hydrochloric acid (Fisher Scientific, A144) and 600 μl of hexane (Fisher Scientific, H302).

Techniques: Targeted Proteomics