Ferroptosis is characterized by the release of lipid mediators. (a) Quantification of a time-dependent release of arachidonic acid metabolites (5-HETE, 11-HETE, 12-HETE and 15-HETE) in the cell culture media after inducible Gpx4 deletion (KO) using LC–MS/MS analysis. (b) Impact of small-molecule inhibitors on HETE release on Gpx4 deletion. Quantification of released arachidonic acid metabolites was performed by LC–MS/MS. Cells were treated with zileuton (10 μM), MK866 (50 μM), PD146176 (1 μM), MJ33 (10 μM) and Fer1 (0.2 μM) at the time of knockout induction. Analysis was performed in conditioned medium collected 72 h after Gpx4 deletion. (c) Quantification of arachidonic acid metabolites released into the medium 24 h after cell death induction by erastin (2.5 μM), RSL3 (0.75 μM), TNFα (10 ng ml−1) and staurosporine (Stauro; 0.2 μM) using HPLC–MS/MS analysis. In the Gpx4−/− cells, HETE levels were determined 72 h after TAM treatment. Data shown represent the mean ± s.d. of n=3 wells of a 6-well plate from an experiment performed independently three times (a,c) or one time (b). ∗P =0.05, ∗∗P =0.01 (one-way ANOVA, followed by Tukey’s multiple comparisons test (a) or Dunnett’s multiple comparison test (b,c)). (d) Impact of arachidonic acid metabolites on the cell death process elicited by Gpx4 loss. HETEs, HpETEs and OxoHETEs (1 μM) were added to WT or KO Pfa1 cells for Gpx4. Compounds were added 36 h after knockout induction and cell viability was measured 12 h thereafter. H2O2 was used at equimolar concentrations. Data shown represent the mean ± s.d. from n = 4 wells (HpETEs) or n = 6 wells (H2O2) of a 96-well plate from a representative experiment performed three times, ∗P = 0.05, ∗∗P = 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test. (e)=Sterol carrier protein-2 (SCP) inhibitors (SCPI) rescue Gpx4−/− cells from ferroptosis in a dose-dependent manner. Inhibitors were added concomitantly to TAM and cell viability was determined 72 h thereafter. Data shown represent the mean ± s.d. of n = 6 wells of a 96-well plate from a representative experiment performed independently two times, P =0.05 (one-way ANOVA).