Fig. 2: Lysosomal membrane permeabilization mediates ferroptotic cell death.

a, b Live-cell fluorescence imaging with acridine orange or fluorescent dextran in Calu-1 cells treated with RSL3 (0.1 µM) ± inhibitors for 3 h. c Time-dependent change of lactate dehydrogenase (LDH) released from Calu-1 cells treated with RSL3 (0.1 µM) ± Lip-1. d Lysosomal Fe²⁺ detected by Lyso-RhoNox. Calu-1 cells were pretreated with deferoxamine (DFO, 50 µM) for 1 h and then labeled with Lyso-RhoNox (1 µM) and Hoechst 33342 for 30 min. Relative intensity per cell is presented. e Intracellular Fe²⁺ detected by FerroOrange. Calu-1 cells were treated with RSL3 (0.1 µM) ± inhibitors for 3 h and then labeled with FerroOrange (1 µM) and Hoechst 33342 for 30 min. Relative intensity per cell is shown. NBD-Pen fluorescence analysis after LMP in Calu-1 cells treated with RSL3 (0.1 µM) ± Lip-1. Scheme (f), live-cell fluorescence imaging (g), and co-staining with ER-Tracker or CellMask Orange Plasma Membrane Stain at 3 and 5 h (h). i Death of Calu-1 cells treated with RSL3 (0.1 µM) ± inhibitors for 24 h. Inhibitors were added either at the same time as RSL3 (0 h) or 1–6 h later. Inhibitors: Lip-1 (1 µM); NBD-Pen (0.3 µM); Lyso-NBD-Pen (Lyso-NP, more polar diastereomer, 0.1 µM). Data are mean ± s.d. (c, d, e, i). Sample size: (c, i) n = 3 independent experiments, (d) n = 21 number of cells present in three different images from each sample, (e) n = 27 (Control), 36 (RSL3), 28 (RSL3 + Lip-1), 31 (RSL3 + NBD-Pen), 34 (RSL3 + Lyso-NBD-Pen) number of cells present in three different images from each sample. Three independent experiments were performed with similar results (d, e). Statistical analysis: p values were calculated by two-way ANOVA with Sidak’s multiple comparison test (c), by the two-tailed unpaired t test (d) or by one-way ANOVA with Tukey–Kramer test (e). Scale bar, (a, h) 10 µm; (b, d, e, g) 20 µm. Source data are provided as a Source Data file.