Fig. 1: Lysosomal iron triggers the oxidation of membrane lipids.
From: Activation of lysosomal iron triggers ferroptosis in cancer
a, Schematic (left), images (middle) and quantification (right) of labelling cLip-1 (1 μM, 1 h) in cells. n = 3 independent experiments in HT-1080 cells. Data are the mean ± s.d. Asc, ascorbate; Lys., lysosome; PFA, paraformaldehyde. b, Kaplan–Meier survival curves of Rosa26-CreERT2;Gpx4f/f mice treated with cLip-1 (10 mg per kg per day by intraperitoneal injection; n = 6 mice per group). Mantel–Cox log-rank test. Arrows indicate the timing of the two tamoxifen injections. Average days of survival are indicated. c, Fluorescence images of labelled cLip-1 in renal proximal tubules of a Rosa26-CreERT2;Gpx4f/f mouse 7 days after tamoxifen treatment. Images representative of n = 2 mice. d, 1H NMR spectra of Lip-1 titrated with FeCl3 (red, 2:1 Lip-1 to iron; green, 1:1 Lip-1 to iron). e, 1H NMR spectra of Lip-1 titrated with FeCl3 and then TFA. The blue reference spectrum is Lip-1 + 3 eq. TFA. f, Putative binding modes of Lip-1 and iron obtained from molecular modelling. g, Cyclic voltammetry of a FeCl3 solution with Lip-1 (left) or DFO (right) (reduction potentials indicated by pink arrows). SCE, saturated calomel electrode. h–k, Data are for HT-1080 cells. h, Flow cytometry of cells treated with the indicated compounds for 15 min and then with the iron(III) probe RPE for 15 min. n = 9 independent experiments. a.u., arbitrary unit; MFI, mean fluorescence intensity. i, Flow cytometry of cells treated with the indicated compounds for 15 min and then with the iron(II) probe HMRhoNox-M for 15 min. n = 8 independent experiments. j, Bodipy-C11 581/591 flow cytometry of cells treated with RSL3 (1 h) and with bafilomycin-A1 (Baf-A1) or hydroxychloroquine (HCQ) 2 h before. n = 7 independent experiments. One-way analysis of variance (ANOVA). k, Fluorescence imaging of Liperfluo in cells treated with RSL3 (1 h). n = 3 independent experiments. Two-sided unpaired t-test. Data are the mean ± s.d. d,e, NMR spectra recorded at 310 K in methanol-d4. For h and i, P values are from two-sided unpaired t-test compared with vehicle. The following concentrations were used: h,i, Lip-1 (10 µM), HCQ (100 µM); h–j, Baf-A1 (75 nM); j, HCQ (10 µM), RSL3 (200 nM); k, RSL3 (1 µM). Box plots show the interquartile range, with centre lines indicating the medians and whiskers indicating the minimum and maximum values. Scale bar, 10 μm (a,c,k).
