Extended Data Fig. 3: Peroxisomes contribute to ferroptosis sensitivity via the ether lipid biosynthesis pathway.
From: Plasticity of ether lipids promotes ferroptosis susceptibility and evasion
a, Schematic of the known functional pathways involved in lipid metabolism and reduction of reactive oxygen species (ROS) in the peroxisome. VLCFA, very-long-chain fatty acids; BCFA, branched-chain fatty acids; DHAP, dihydroxyacetone phosphate; AGPS, alkylglycerone phosphate synthase. b, Volcano plots showing the lipidomic analysis of 786-O cells expressing sgNC or PEX3-targeting sgRNAs. n = 3 biologically independent samples. Two tailed Student’s t-test. Multiple-testing adjustment was performed using the Benjamini–Hochberg method. c, Viability curves of OVCAR-8 cells expressing negative control (sgNC), FAR1-targeting sgRNAs (left) or AGPS-targeting sgRNAs (right) and treated with indicated concentrations of RSL3 for 72 h. n = 3 biologically independent samples. Representative results of experiment performed in triplicate. d, Fluorescent imaging showing nuclear staining by Hoechst 33342 in OVCAR-8 cells with the indicated genetic perturbations and treated with vehicle (DMSO) or indicated concentrations of ML210 for 5 days. Representative images from experiment performed once, and each condition has three biological replicates. e, Relative growth rates measured by areas of live-cell coverage in OVCAR-8 cells expressing sgNC, AGPS sg2 or FAR1 sg2 and treated with indicated concentrations of ML210 for 5 days. n = 2 or 3 biologically independent samples. Data of experiment performed once. f, Immunoblot analysis of AGPS levels in 786-O cells expressing negative control (sgNC) or AGPS-targeting sgRNAs. g, Viability curves of 786-O cells expressing sgNC or AGPS-targeting sgRNAs treated with indicated concentrations of ML210 or RSL3 for 48 h. n = 4 biologically independent samples. Representative result of experiment performed in triplicate. h, Immunoblot analysis of FAR1 levels in HuH-7 cells expressing negative control (sgNC) or AGPS-targeting sgRNAs. β-Actin was used as a loading control. i, Viability curves of 786-O cells expressing sgNC or FAR1-targeting sgRNAs treated with indicated concentrations of ML210 or RSL3 for 48 h. n = 4 biologically independent samples. Representative result of experiment performed in triplicate. j, Immunoblot showing AGPS levels in OVCAR-8 cells expressing sgNC or AGPS sg2, and AGPS−/− single cell clone (SCC)#9. k, Viability curves of OVCAR-8 cells expressing sgNC or AGPS sg2, and AGPS−/− SCC9 treated with ML210 for 72 h. n = 3 biologically independent samples. Representative result of experiment performed in duplicate. l, Immunoblot showing FAR1 levels in OVCAR-8 cells expressing sgNC or FAR1 sg1, and FAR1−/− single cell clone (SCC)#9. m, Viability curves of OVCAR-8 cells expressing sgNC or FAR1 sg1, and FAR1−/− SCC9 treated with ML210 for 72 h. n = 3 biologically independent samples. Representative result of experiment performed in duplicate. Immunoblots are representative data of experiments performed twice. See Supplementary Information for uncropped immunoblot images. β-Actin or GAPDH was used as a loading control. For viability curves, data are mean ± s.d.
