Fig. 5

Therapy-induced lipid remodeling leads to increased membrane PUFA levels and lipid peroxidation and underpins GPX4 dependence. a LNCaP cells were treated for the indicated times with Enz (10 μM), and lipids were analyzed by LC/MS. Individual sum compositions of phosphatidylethanolamine are shown (n = 2, mean ± SD). b LNCaP cells were treated as in a, and membrane fluidity was measured by qSCI based on the mean fluorescence intensity (MFI) of Di-4-ANEPPDHQ (n = 8 wells, ~ 750 cells/well, mean ± SD, representative results from 2 independent experiments). c Relative mRNA expression changes of indicated lipid remodeling enzymes (elongases and desaturases) were calculated from the microarray data described in Fig. 2a. d LNCaP cells were treated for the indicated times with Enz (10 μM), and lipid peroxidation was measured by qSCI based on the mean fluorescence intensity (MFI) of Bodipy-C11 (n > 9000 cells, mean ± SD, representative results from 2 independent experiments). e LNCaP cells were cultured in growth media without (D0) or with Enz (10 μM, D14) for 14 days including the final 24 h in the presence of DMSO (0) or the indicated concentrations (0.5–1.5 μM) of GPX4 inhibitor RSL3. Lipid peroxide scavenger Trolox (100 μM) and ferroptosis inhibitor Ferrostatin-1 (10 μM) were used as controls to demonstrate cell death through ferroptosis. The percentage of dead cells was calculated based on qSCI of Hoechst 3342 (total cell count) and propidium iodide (dead cells) staining (n = 3 wells/treatment with > 4000 cells/well, representative result of 3 independent experiments). f Relative mRNA expression changes of indicated key factors of ferroptosis and glutathione homeostasis were calculated from the microarray data described in Fig. 2a. g LNCaP cells seeded in a 96-well plate were co-treated with the indicated compounds (orlistat 10 μM, CAY10499 17.5 μM, SC26196 30 μM, TOFA 2.5 μM, fatostatin 5 μM, arachidonic acid (AA) 20 μM, Trolox 100 μM) in the absence (FBS) or presence of Enz (10 μM, FBS+ENZ), and cell confluence was measured every 2 h by live cell imaging with an IncuCyte system (n = 3, mean ± SD). The gray dotted line indicates the time point of media change. After 7 days of co-treatment, cells were treated with vehicle control (DMSO/FBS) or RSL3 (1.25 μM) to assess GPX4 dependence by monitoring changes to cell confluence for another day. The red dotted line indicates the start of RSL3 treatment. Cell death was then measured by qSCI (bottom graph) as described in (e; n = 3 wells/treatment with > 4000 cells/well, representative result of 2 independent experiments). Statistical analysis: a two-way ANOVA followed by Tukey’s multiple comparisons test relative to D0; b–f one-way ANOVA followed by Dunnett’s multiple comparisons test compared to D0 or FBS+RSL3 (g, light brown labels) and FBS+ENZ+RSL3 (g, blue labels); ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001