Right panels: formulas of oxygenated SAPE and detected fragments (red). Left panels: MS 2 and MS 3 (inserts) fragmentation of di- (m/z 798.5300) and tri-oxygenated (m/z 814.5262) SAPE.
( c) LC-MS identification of 15-LOX induced SAPE oxidation products in cell lysates. Data are mean±s.d., n=3 *, # indicate P < 0.05 vs. Without RS元, SAPE-OOH (0.9 μM) did not induce ferroptosis ( b, insert). ( a) Effects of AA-OOH (2.5 μM), AA-OOH-CoA (2.5 μM), and ( b) SAPE-OOH on RS元 (100 nM, 6 hrs) triggered ferroptosis in WT or Acsl4 KO cells.
( g) Distribution of free and esterified PUFA-OOH in WT and Acsl4 KO Pfa1 cells treated with RS元 (100 nM, 6 hrs).
( f) Contents of AA-CoA and AdA-CoA in WT and Acsl4 KO cells. ( e) ACSL inhibitor, Triacsin C, suppresses ferroptosis in Pfa1 cells. LiperFluo (green, both panels, scale bars 5 μm). ( d) Fluorescence responses from Mito-FAP (red, upper panel) and ER-FAP (red, lower panel) vs. ( c) WT and Acsl4 KO cells were treated with RS元 (100 nM) for 2, 4, and 6 hrs before cell death analysis. For the statistical analysis, each stage position counted as one data entry. The data were from a minimum of 10 stage positions. Insert: Fluorescence time course after RS元 treatment (100 nM) in WT and Acsl4 KO cells with a time control. ( b) LiperFluo fluorescence intensity following RS元 treatment in WT and Acsl4 KO cells. ( a) Live cell fluorescence imaging of lipid hydroperoxides in WT and Acsl4 KO cells treated with RS元 (100 nM, 6 hrs, scale bar 5 μm). This oxidative PE death pathway may also represent a target for drug discovery. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA).