(c) WT and Acsl4 KO cells were treated with RSL3 (100 nM) for 2, 4, and 6 hrs before cell loss of life analysis

(c) WT and Acsl4 KO cells were treated with RSL3 (100 nM) for 2, 4, and 6 hrs before cell loss of life analysis. varieties which become death indicators while tocopherols and tocotrienols suppress LOX and drive back ferroptosis recommending an unexpected homeostatic physiological part of supplement E. This oxidative PE death pathway may represent a target for drug discovery also. < 0.05 WT+RSL3 cells (t-test). (c) WT and Acsl4 KO cells had been treated with RSL3 (100 nM) for 2, 4, and 6 hrs before cell loss of life evaluation. Data are mean s.d., n=3. *< 0.05 ACSL4 KO+RSL3 (t-test). (d) Fluorescence reactions from Mito-FAP (reddish colored, upper -panel) and ER-FAP (reddish colored, lower -panel) LiperFluo (green, both sections, scale pubs 5 m). (e) ACSL inhibitor, Triacsin C, suppresses ferroptosis in Pfa1 cells. AA (2.5 M,16 hrs); RSL3 (100 nM, 6 hrs); Triacsin C (2.5 M, 6 hrs). Data are mean s.d., n=3. *< 0.05 control (t-test). (f) Material of AA-CoA and AdA-CoA in WT and Acsl4 KO cells. Data are mean s.d., n=3. *< 0.05 WT cells (t-test). (g) Distribution of free of charge and esterified PUFA-OOH in WT and Acsl4 KO Pfa1 cells treated with RSL3 (100 nM, 6 hrs). Measurements of reactive air varieties (ROS) and pro-oxidant activity towards non-lipidic fluorogenic substrates (e.g., a lipid ROS probe, C11-BODIPY 581/591, or linoleamide alkyne click-conjugated by cyclo-addition response with fluorescein azide) have already been utilized mainly because surrogate procedures for lipid peroxidation9. While fluorescence reactions from these probes demonstrated general activation during ferroptosis, they cannot reveal the immediate creation of lipid hydroperoxides. Both C11-BODIPY and LiperFluo can react with peroxyl radicals whereas LiperFluo (however, not C11-BODIPY) interacts with (phospho)lipid hydroperoxides10. In comparison, LiperFluo fluorescence reviews intracellular sites of lipid hydroperoxide accumulation8 reliably. GPX4 decreases hydroperoxides of polyunsaturated essential fatty acids (PUFA-OOH) and phospholipids (PL-OOH)7. Esterification of PUFA into phospholipids needs acyl-CoA synthase catalyzed development of PUFA-CoA. Particularly, ACSL4 catalyzes synthesis of long-chain polyunsaturated-CoAs having a choice for AA11, facilitating their esterification into phospholipids12 thus. While hereditary ablation13 or inhibition of ACSL4 by Triacsin C had been both effective in avoiding RSL3 induced cell loss of life (Fig. 1e) we found out better quality LiperFluo fluorescence response from Acsl4 KO cells in comparison to WT cells (Fig. 1a, b). Because Acsl4 KO cells possess reduced degrees of polyunsaturated-acyl-CoAs (Fig. 1f), they most likely accumulate free of charge PUFA-OOH (instead of esterified PL-OOH) leading to raised LiperFluo fluorescence emission. To check this, we performed LC-MS/MS analysis of free of charge PL-OOH and PUFA-OOH in WT Acsl4 KO cells. This was attained by the usage of platelet-activating element acetylhydrolase (PAF-AH), an enzyme specifically cleaving the oxidized PUFA residues from phospholipids14 to produce lyso-phospholipids and FA-OOH. Certainly, in Acsl4 KO cells, RSL3 induced mainly accumulation of free of charge oxygenated PUFA (Fig. 1g) - as opposed to higher degrees of esterified oxygenated AA and adrenic acidity (AdA, C22:4) in WT cells (Fig. 1g). Assessments from the response price constants for AA-OOH and purified PE-OOH with LiperFluo in ethanol demonstrated that its reactivity towards free of Olaquindox charge PUFA-OOH was somewhat greater than with PL-OOH using the response rate constants of just one 1.60.1103M?1s?1 15 and 1.20.1103M?1s?1, respectively. Therefore higher material of free of charge PUFA-OOH and their higher reactivity toward LiperFluo both added to the solid fluorescence response to LiperFluo in Ascl4 KO cells. AA enhances ferroptotic response in RSL3-treated cells Recommending that esterified oxygenated PUFA, become the proximate executioners of ferroptotic loss of life, we supplemented Acsl4 and WT KO cells with exogenous AA. This led to a 24% boost of ferroptosis in RSL3-treated WT cells in support of a 13% boost of loss of life in Acsl4 KO cells (Fig. 2a). Appropriately, LC-MS/MS evaluation (after PAF-AH treatment) proven higher build up of esterified oxygenated AA in phospholipids of WT vs Acsl4 KO cells pursuing RSL3 treatment (Fig. 1g). Additionally, we noticed that supplementation with AA activated elongation activity leading to the increased content material of AdA and its own oxygenated forms (Fig. 2b, c). The levels of oxygenated.Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 works as a particular anti-ferroptotic save pathway. tocotrienols suppress LOX and drive back ferroptosis recommending an unexpected homeostatic physiological part of supplement E. This oxidative PE loss of life pathway could also represent a focus on for drug finding. < 0.05 WT+RSL3 cells (t-test). (c) WT and Acsl4 KO cells had been treated with RSL3 (100 nM) for 2, 4, and 6 hrs before cell loss of life evaluation. Data are mean s.d., n=3. *< 0.05 ACSL4 KO+RSL3 (t-test). (d) Fluorescence reactions from Mito-FAP (reddish colored, upper -panel) and ER-FAP (reddish colored, lower -panel) LiperFluo (green, both sections, scale pubs 5 m). (e) ACSL inhibitor, Triacsin C, suppresses ferroptosis in Pfa1 cells. AA (2.5 M,16 hrs); RSL3 (100 nM, 6 hrs); Triacsin C (2.5 M, 6 hrs). Data are mean s.d., n=3. *< 0.05 control (t-test). (f) Material of AA-CoA and AdA-CoA in WT and Acsl4 KO cells. Data are mean s.d., n=3. *< 0.05 WT cells (t-test). (g) Distribution of free of charge and esterified PUFA-OOH in WT and Acsl4 KO Pfa1 cells treated with RSL3 (100 nM, 6 hrs). Measurements of reactive air varieties (ROS) and pro-oxidant activity towards non-lipidic fluorogenic substrates (e.g., a lipid ROS probe, C11-BODIPY 581/591, or linoleamide alkyne click-conjugated by cyclo-addition response with fluorescein azide) have already been utilized mainly because surrogate procedures for lipid peroxidation9. While fluorescence reactions from these probes demonstrated general activation during ferroptosis, they cannot reveal the immediate creation of lipid hydroperoxides. Both C11-BODIPY and LiperFluo can react with peroxyl radicals whereas LiperFluo (however, not C11-BODIPY) interacts with (phospho)lipid hydroperoxides10. In comparison, LiperFluo fluorescence reliably reviews intracellular sites of lipid hydroperoxide build up8. GPX4 decreases hydroperoxides of polyunsaturated essential fatty acids (PUFA-OOH) and phospholipids (PL-OOH)7. Esterification of PUFA into phospholipids needs acyl-CoA synthase catalyzed development of PUFA-CoA. Particularly, ACSL4 catalyzes synthesis of long-chain polyunsaturated-CoAs having a choice for AA11, therefore facilitating their esterification into phospholipids12. While hereditary ablation13 or inhibition of ACSL4 by Triacsin C had been both effective in avoiding RSL3 induced cell loss of life (Fig. 1e) we present better quality LiperFluo fluorescence response from Acsl4 KO cells in comparison to WT cells (Fig. 1a, b). Because Acsl4 KO cells possess reduced degrees of polyunsaturated-acyl-CoAs (Fig. 1f), they most likely accumulate free of charge PUFA-OOH (instead of esterified PL-OOH) leading to raised LiperFluo fluorescence emission. To check this, we performed LC-MS/MS evaluation of free of charge PUFA-OOH and PL-OOH in WT Acsl4 KO cells. This is achieved by the usage of platelet-activating aspect acetylhydrolase (PAF-AH), an enzyme particularly cleaving the oxidized PUFA residues from phospholipids14 to produce FA-OOH and lyso-phospholipids. Certainly, in Acsl4 KO cells, RSL3 induced mostly accumulation of free of charge oxygenated PUFA (Fig. 1g) - as opposed to higher degrees of esterified oxygenated AA and adrenic acidity (AdA, C22:4) in WT cells (Fig. 1g). Assessments from the response price constants for AA-OOH and purified PE-OOH with LiperFluo in ethanol demonstrated that its reactivity towards free of charge PUFA-OOH was somewhat greater than with PL-OOH using the response rate constants of just one 1.60.1103M?1s?1 15 and 1.20.1103M?1s?1, respectively. Hence higher items of free of charge PUFA-OOH and their higher reactivity toward LiperFluo both added to the sturdy fluorescence response to LiperFluo in Ascl4 KO cells. AA enhances ferroptotic response in RSL3-treated cells Recommending that esterified oxygenated PUFA, become the proximate executioners of ferroptotic loss of life, we supplemented WT and Acsl4 KO cells with exogenous AA. This led to a 24% boost of ferroptosis in RSL3-treated WT cells in support of a 13% boost of loss of life in Acsl4 KO cells (Fig. 2a). Appropriately, LC-MS/MS evaluation (after PAF-AH treatment) showed higher deposition of esterified oxygenated AA in phospholipids of WT vs Acsl4 KO cells pursuing RSL3 treatment (Fig. 1g). Additionally, we noticed that supplementation with AA prompted elongation activity leading to the increased articles of AdA and its own oxygenated forms (Fig. 2b, c). The levels of oxygenated esterified.Cas9-expressing mouse embryonic fibroblasts (Pfa1) were transfected using a plasmid expressing a gRNA targeting exon 1 of the Acsl4 gene. particular anti-ferroptotic recovery pathway. Lipoxygenases (LOX) generate doubly- and triply-oxygenated (15-hydroperoxy)-di-acylated PE types which become death indicators while tocopherols and tocotrienols suppress LOX and drive back ferroptosis recommending an unexpected homeostatic physiological function of supplement E. This oxidative PE loss of life pathway could also represent a focus on for drug breakthrough. < 0.05 WT+RSL3 cells (t-test). (c) WT and Acsl4 KO cells had been treated with RSL3 (100 nM) for 2, 4, and 6 hrs before cell loss of life evaluation. Data are mean s.d., n=3. *< 0.05 ACSL4 KO+RSL3 (t-test). (d) Fluorescence replies from Mito-FAP (crimson, upper -panel) and ER-FAP (crimson, lower -panel) LiperFluo (green, both sections, scale pubs 5 m). (e) ACSL inhibitor, Triacsin C, suppresses ferroptosis in Pfa1 cells. AA (2.5 M,16 hrs); RSL3 (100 nM, 6 hrs); Triacsin C (2.5 M, 6 hrs). Data are mean s.d., n=3. *< 0.05 control (t-test). (f) Items of AA-CoA and AdA-CoA in WT and Acsl4 KO cells. Data are mean s.d., n=3. *< 0.05 WT cells (t-test). (g) Distribution of free of charge and esterified PUFA-OOH in WT and Acsl4 KO Pfa1 cells treated with RSL3 (100 nM, 6 hrs). Measurements of reactive air types (ROS) and pro-oxidant activity towards non-lipidic fluorogenic substrates (e.g., a lipid ROS probe, C11-BODIPY 581/591, or linoleamide alkyne click-conjugated by cyclo-addition response with fluorescein azide) have already been utilized simply because surrogate methods for lipid peroxidation9. While fluorescence replies from these probes demonstrated general activation during ferroptosis, they cannot reveal the immediate creation of lipid hydroperoxides. Both C11-BODIPY and LiperFluo can react with peroxyl radicals whereas LiperFluo (however, not C11-BODIPY) interacts with (phospho)lipid hydroperoxides10. In comparison, LiperFluo fluorescence reliably reviews intracellular sites of lipid hydroperoxide deposition8. GPX4 decreases hydroperoxides of polyunsaturated essential fatty acids (PUFA-OOH) and phospholipids (PL-OOH)7. Esterification of PUFA into phospholipids needs acyl-CoA synthase catalyzed development of PUFA-CoA. Particularly, ACSL4 catalyzes synthesis of long-chain polyunsaturated-CoAs using a choice for AA11, hence facilitating their esterification into phospholipids12. While hereditary ablation13 or inhibition of ACSL4 by Triacsin C had been both effective in avoiding RSL3 induced cell loss of life (Fig. 1e) we present better quality LiperFluo fluorescence response from Acsl4 KO cells in comparison to WT cells (Fig. 1a, b). Because Acsl4 KO cells possess reduced degrees of polyunsaturated-acyl-CoAs (Fig. 1f), they most likely accumulate free of charge PUFA-OOH (instead of esterified PL-OOH) leading to raised LiperFluo fluorescence emission. To check this, we performed LC-MS/MS evaluation of free of charge PUFA-OOH and PL-OOH in WT Acsl4 KO cells. This is achieved by the usage of platelet-activating aspect acetylhydrolase (PAF-AH), an enzyme particularly cleaving the oxidized PUFA residues from phospholipids14 to produce FA-OOH and lyso-phospholipids. Certainly, in Acsl4 KO cells, RSL3 induced mostly accumulation of free of charge oxygenated PUFA (Fig. 1g) - as opposed to higher degrees of esterified oxygenated AA and adrenic acidity (AdA, C22:4) in WT cells (Fig. 1g). Assessments from the response price constants for AA-OOH and purified PE-OOH with LiperFluo in ethanol demonstrated that its reactivity towards free of charge PUFA-OOH was somewhat greater than with PL-OOH using the response rate constants of just one 1.60.1103M?1s?1 15 and 1.20.1103M?1s?1, respectively. Hence higher items of free of charge PUFA-OOH and their higher reactivity toward LiperFluo both added to the sturdy fluorescence response to LiperFluo in Ascl4 KO cells. AA enhances ferroptotic response in RSL3-treated cells Recommending that esterified oxygenated PUFA, become the proximate executioners of ferroptotic loss of life, we supplemented WT and Acsl4 KO cells with exogenous AA. This led to a 24% boost of ferroptosis in RSL3-treated WT cells in support of a 13% boost of loss of life in Acsl4 KO cells (Fig. 2a). Appropriately, LC-MS/MS evaluation (after PAF-AH treatment) showed higher deposition of esterified oxygenated AA in phospholipids of WT vs Acsl4 KO cells pursuing RSL3 treatment (Fig. 1g). Additionally, we noticed that supplementation with AA prompted elongation activity leading to the increased articles of AdA and its own oxygenated forms (Fig. 2b, c). The levels of oxygenated esterified AA and AdA had been low in RSL3-treated Acsl4 KO cells than in RSL3-treated WT cells (72.227.0 and 28.28.0 in comparison to 199.326.2 and 137.877.7 pmol/mol phospholipids, respectively, < 0.05 control, RSL3 (or Acsl4 KO+RSL3), RSL3+AA, respectively (t-test). (b) LC-MS structured high temperature maps for oxygenated esterified AA (C20:4) and AdA (C22:4) (normalized to matching WT group, in dark), displaying their relative adjustments after different remedies. (c) Schema of metabolic pathways for C20:4, C22:4 and their oxygenated items. (d) Degrees of oxidized AA and AdA esterified into phospholipids.Isocratic cellular phase includes acetonitrile/water/triethylamine/acetic acid solution - 900/100/5/5 v/v was employed for separation. ferroptosis recommending an unexpected homeostatic physiological function of supplement E. This oxidative PE loss of life pathway could also represent a focus on for drug breakthrough. < 0.05 WT+RSL3 cells (t-test). (c) WT and Acsl4 KO cells had been treated with RSL3 (100 nM) for 2, 4, and 6 hrs before cell loss of life evaluation. Data are mean s.d., n=3. *< 0.05 ACSL4 KO+RSL3 (t-test). (d) Fluorescence replies from Mito-FAP (crimson, upper -panel) and ER-FAP (crimson, lower -panel) LiperFluo (green, both sections, scale pubs 5 m). (e) ACSL inhibitor, Triacsin C, suppresses ferroptosis in Pfa1 cells. AA (2.5 M,16 hrs); RSL3 (100 nM, 6 hrs); Triacsin C (2.5 M, 6 hrs). Data are mean s.d., n=3. *< 0.05 control (t-test). (f) Items of AA-CoA and AdA-CoA in WT and Acsl4 KO cells. Data are mean s.d., n=3. *< 0.05 WT cells (t-test). (g) Distribution of free of charge and esterified PUFA-OOH in WT and Acsl4 KO Pfa1 cells treated with RSL3 (100 nM, 6 hrs). Measurements of reactive air types (ROS) and pro-oxidant activity towards non-lipidic fluorogenic substrates (e.g., a lipid ROS probe, C11-BODIPY 581/591, or linoleamide alkyne click-conjugated by cyclo-addition response with fluorescein azide) have already been utilized simply because surrogate methods for lipid peroxidation9. While fluorescence replies from these probes demonstrated general activation during ferroptosis, they cannot reveal the immediate creation of lipid hydroperoxides. Both C11-BODIPY and LiperFluo can react with peroxyl radicals whereas LiperFluo (however, not C11-BODIPY) interacts with (phospho)lipid hydroperoxides10. In comparison, LiperFluo fluorescence reliably reviews intracellular sites of lipid hydroperoxide deposition8. GPX4 decreases hydroperoxides of polyunsaturated essential fatty acids (PUFA-OOH) and phospholipids (PL-OOH)7. Esterification of PUFA into phospholipids needs acyl-CoA synthase catalyzed development of PUFA-CoA. Particularly, ACSL4 catalyzes synthesis of long-chain polyunsaturated-CoAs using a choice for AA11, hence facilitating their esterification into phospholipids12. While hereditary ablation13 or inhibition Olaquindox of ACSL4 by Triacsin C had been both effective in avoiding RSL3 induced cell loss of life (Fig. 1e) we present better quality LiperFluo fluorescence response from Acsl4 KO cells in comparison to WT cells (Fig. 1a, b). Because Acsl4 KO cells possess reduced degrees of polyunsaturated-acyl-CoAs (Fig. 1f), they most likely accumulate free of charge PUFA-OOH (instead of esterified PL-OOH) leading to raised LiperFluo fluorescence emission. To check this, we performed LC-MS/MS evaluation of free of charge PUFA-OOH and PL-OOH in WT Acsl4 KO cells. This is achieved by the usage of platelet-activating aspect acetylhydrolase (PAF-AH), an enzyme particularly cleaving the oxidized PUFA residues from phospholipids14 to produce FA-OOH and lyso-phospholipids. Certainly, in Acsl4 KO cells, RSL3 induced mostly accumulation of free of charge oxygenated PUFA (Fig. 1g) - as opposed to higher degrees of esterified oxygenated AA and adrenic acidity (AdA, C22:4) in WT cells (Fig. 1g). Assessments from the response price constants for AA-OOH and purified PE-OOH with LiperFluo in ethanol demonstrated that its reactivity towards free of charge PUFA-OOH was somewhat greater than with PL-OOH using the response rate constants of just one 1.60.1103M?1s?1 15 and 1.20.1103M?1s?1, respectively. Hence higher items of free of charge PUFA-OOH and their higher reactivity toward LiperFluo both added to the sturdy fluorescence response to LiperFluo in Ascl4 KO cells. AA enhances ferroptotic response in RSL3-treated cells Recommending that esterified oxygenated PUFA, become the proximate executioners of ferroptotic loss of life, we supplemented WT and Acsl4 KO cells with exogenous AA. This led to a 24% boost of ferroptosis in RSL3-treated WT cells in support of a 13% boost of loss of life in Acsl4 KO cells (Fig. 2a). Appropriately, LC-MS/MS evaluation (after PAF-AH treatment) showed higher deposition of esterified oxygenated AA in phospholipids of WT vs Acsl4 KO cells pursuing RSL3 treatment (Fig. 1g). Additionally, we noticed that supplementation with AA prompted elongation activity leading to the increased articles of AdA and its own oxygenated forms (Fig. 2b, c). The levels of oxygenated esterified AA and AdA had been low in RSL3-treated Acsl4 KO cells than in RSL3-treated WT cells (72.227.0 and 28.28.0 in comparison to 199.326.2 and 137.877.7 pmol/mol phospholipids, respectively, < 0.05 control, RSL3 (or Acsl4 KO+RSL3), RSL3+AA, respectively (t-test). (b) LC-MS structured high temperature maps for oxygenated esterified AA (C20:4) and AdA (C22:4) (normalized to matching WT group, in dark), displaying their relative adjustments after different remedies. (c) Schema of metabolic pathways for C20:4, C22:4 and their oxygenated items. (d) Degrees of oxidized AA and AdA esterified into phospholipids in WT and Acsl4 KO Pfa1 cells. Data are mean s.e.m., n=3. *< 0.05 WT cells (one-tailed t-test). (e) Lpcat3 KD reduced RSL3-induced ferroptosis in MLE cells..Simply no changes in this content of alkyl- or alkenyl-PE types were discovered (Fig. and 6 hrs before cell loss of life evaluation. Data are mean s.d., n=3. *< 0.05 ACSL4 KO+RSL3 (t-test). (d) Fluorescence replies from Mito-FAP (crimson, upper -panel) and ER-FAP (crimson, lower -panel) LiperFluo (green, both sections, scale pubs 5 m). (e) ACSL inhibitor, Triacsin C, suppresses ferroptosis in Pfa1 cells. AA (2.5 M,16 hrs); RSL3 (100 nM, 6 hrs); Triacsin C (2.5 M, 6 hrs). Rabbit polyclonal to AFG3L1 Data are mean s.d., n=3. *< 0.05 control (t-test). (f) Items of AA-CoA and AdA-CoA in WT and Acsl4 KO cells. Data are mean s.d., n=3. *< 0.05 WT cells (t-test). (g) Distribution of free of charge and esterified PUFA-OOH in WT and Acsl4 KO Pfa1 cells treated with RSL3 (100 nM, 6 hrs). Measurements of reactive air types (ROS) and pro-oxidant activity towards non-lipidic fluorogenic substrates (e.g., a lipid ROS probe, C11-BODIPY 581/591, or linoleamide alkyne click-conjugated by cyclo-addition response with fluorescein azide) have been utilized as surrogate measures for lipid peroxidation9. While fluorescence responses from these probes showed overall activation during ferroptosis, they could not reveal the direct production of lipid hydroperoxides. Both C11-BODIPY and LiperFluo can react with peroxyl radicals whereas LiperFluo (but not C11-BODIPY) interacts with (phospho)lipid hydroperoxides10. By contrast, LiperFluo fluorescence reliably reports intracellular sites of lipid hydroperoxide accumulation8. GPX4 reduces hydroperoxides of polyunsaturated fatty acids (PUFA-OOH) and phospholipids (PL-OOH)7. Esterification of PUFA into phospholipids requires acyl-CoA synthase catalyzed formation of PUFA-CoA. Specifically, ACSL4 catalyzes synthesis of long-chain polyunsaturated-CoAs with a preference for AA11, thus facilitating their esterification into phospholipids12. While genetic ablation13 or inhibition of ACSL4 by Triacsin C were both effective in protecting against RSL3 induced cell death (Fig. 1e) we found more robust LiperFluo fluorescence response from Acsl4 KO cells compared to WT cells (Fig. 1a, b). Because Acsl4 KO cells have decreased levels of polyunsaturated-acyl-CoAs (Fig. 1f), they likely accumulate free PUFA-OOH (rather than esterified PL-OOH) causing elevated LiperFluo fluorescence emission. To test this, we performed LC-MS/MS analysis of free PUFA-OOH and PL-OOH in WT Acsl4 KO cells. This was achieved by the use of platelet-activating factor acetylhydrolase (PAF-AH), an enzyme specifically cleaving the oxidized PUFA residues from phospholipids14 to yield FA-OOH and lyso-phospholipids. Indeed, in Acsl4 KO cells, RSL3 induced predominantly accumulation of free oxygenated PUFA (Fig. 1g) - in contrast to higher levels of esterified oxygenated AA and adrenic acid (AdA, C22:4) in WT cells (Fig. 1g). Assessments of the reaction rate constants for AA-OOH and purified PE-OOH with LiperFluo in ethanol showed that its reactivity towards free PUFA-OOH was slightly higher than with PL-OOH with the reaction rate constants of 1 1.60.1103M?1s?1 15 and 1.20.1103M?1s?1, respectively. Thus higher contents of free PUFA-OOH and their higher reactivity toward LiperFluo both contributed to the robust fluorescence response to LiperFluo in Ascl4 KO cells. AA enhances ferroptotic response in RSL3-treated cells Suggesting that esterified oxygenated PUFA, act as the proximate executioners of ferroptotic death, we supplemented WT and Acsl4 KO Olaquindox cells with exogenous AA. This resulted in a 24% increase of ferroptosis in RSL3-treated WT cells and only a 13% increase of death in Acsl4 KO cells (Fig. 2a). Accordingly, LC-MS/MS analysis (after PAF-AH treatment) exhibited higher accumulation of esterified oxygenated AA.