Fig. 4

HILPDA enriches polyunsaturated lipids and promotes ferroptosis sensitivity downstream of HIF-2α. a Scheme summarizing the experimental strategy for identifying the HIF-2α target genes mediating ferroptosis susceptibility in 786-O cells. b Heatmap showing the RNA-Seq analysis of WT 786-O cells and three EPAS1^−/− clones (1D1, 1D7, and 1E3). HIF-2α-dependent known lipid metabolism genes are highlighted. c Viability curves of cDNA screening results in EPAS1^−/− clones treated with a 7-point, 2-fold dilution series of ML210 or RSL3. Viability is relative to that of each cell line under zero ML210 or RSL3 treatment, respectively. n = 4, error bars are omitted for visual clarity. d Viability curves of HILPDA, G0S2, or EGFP-overexpressing EPAS1^−/− 1D1 and 1D7 cells treated with ML210 or RSL3 for 48 h. Viability is relative to the respective DMSO-treated conditions. n = 4, Representative plot of experiments repeated three times. e qRT-PCR analysis of HILPDA mRNA levels in 786-O cells expressing shNC or shHILPDAs. B2M was used as an internal control. n = 3. Student’s T-test. *p < 0.05. f Immunoblot analysis of HILPDA protein levels in 786-O cells expressing shNC or shHILPDAs. β-Actin was used as a loading control. g Viability curves for 786-O cells expressing shNC or two most effective shHILPDAs under indicated concentrations of ML210 or RSL3. Viability is relative to the DMSO-treated conditions, n = 4. Representative plot of experiments repeated three times. h Volcano plots showing the changes in each TAG (left panels) and PE/ePE (right panels) species grouped as PUFA-containing (red fill) or SFA/MUFA-only (white fill) lipids between the indicated cell lines. n = 3. i Bar graph showing the relative abundances of the PUFA-PE/ePEs in the conditions tested. n = 3. S, ferroptosis-sensitive; (R), ferroptosis-resistant. j Lipid droplet abundances analyzed by flow cytometry quantitation of BODIPY-493/503 signal in 1D1 and 1D7 EPAS1^−/− cells expressing exogenous EGFP, HILPDA, G0S2 or PLIN2. Representative plot of experiments repeated three times. k Scheme summarizing the molecular network driving the intrinsic GPX4 dependency and ferroptosis susceptibility in clear-cell carcinomas. Abbreviations: PUFA, polyunsaturated fatty acids, e.g. arachidonic acid (C20:4); TAG, triacylglycerols; PE, phosphatidylethanolamine; ePE, vinyl ether-linked PE-plasmalogens. Metabolites highlighted in red indicate promoters of ferroptosis susceptibility. Error bars: ±s.d