Extended Data Fig. 5: Fento-1 induces the oxidation of phospholipids and cell death by activating lysosomal iron (Part 1).
From: Activation of lysosomal iron triggers ferroptosis in cancer
a, Fluorescence imaging of Fento-1, marmycin and the cell surface marker CD44 in cells treated at 4 °C for 1 h. Scale bar, 10 μm. n = 3 independent experiments. Data are mean ± s.d. b, Fluorescence imaging of Fento-1 in BacMam transduced cells of GFP-labelled cell organelle markers. Scale bar, 10 μm. n = 3 independent experiments. Data are mean ± s.d. c, Bodipy-C11 581/591 flow cytometry of cells treated with Fento-1, marmycin and cCW alone or in combination for 6 h. Representative of n = 3 independent experiments. d, Viability of cells treated for 6 h. Data are mean ± s.e.m. n = 3 independent experiments. e, Viability of cells treated for 72 h. Data are mean ± s.e.m. n = 3 independent experiments. f, Lipidomics of oxidised phospholipids in cells treated with Fento-1 or ferroptosis inducers for 24 h. n = 5 independent experiments. g, Lipidomics of oxidised phospholipids in cells treated with Fento-1. n = 4 independent experiments. Full heatmap, Supplementary Information. h, Lipidomics of oxidised phospholipids of lysosomal extracts in cells treated with Fento-1 (10 µM, 1 h). n = 3 independent experiments. i, Western blot of proteins from cells treated with Fento-1, marmycin, cCW or marmycin + cCW. γ-tubulin is a sample loading control. Representative of n = 3 independent experiments. j, Western blot of proteins from cells treated with Fento-1, marmycin, cCW or marmycin + cCW (48 h). γ-tubulin is a sample processing control. Representative of n = 2 independent experiments with similar results. k, Bodipy-C11 581/591 flow cytometry of cells treated with Fento-1 (24 h) and inhibitors used 2 h prior. n = 7 independent experiments. Box plots: interquartile range, centre lines = medians and whiskers = the minimum and maximum values. l, Fluorescence imaging of 4-HNE treated with Fento-1 (1 h). Two-sided unpaired t-test. n = 3 independent experiments. Data are mean ± s.d. Scale bar, 10 μm. m, Quantification of glycerol in cells treated with Fento-1 (24 h). Data are mean ± s.e.m. n = 5 independent experiments. Concentrations used unless stated otherwise: Fento-1 (1 µM), marmycin (1 µM), cCW (1 µM), erastin (10 µM), RSL3 (100 nM), iFSP1 (10 µM), Toc (100 µM), Def (100 µM), Lip-1 (1 µM) and cLip-1 (1 µM). k, m, Kruskal–Wallis test with Dunn’s post-test. Phosphatidylcholine (PC) lipids displayed in the main figures. Phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), Supplementary Information.
