Fig. 4.

The cytotoxic mechanism of SCs is independent from major cellular pathways regulating ferroptosis. A. Labile iron content of MDA-MB-231 cells (1 × 10⁶/25 cm²) treated with vehicle (‘w/o’, 0.1 % DMSO), erastin (3 μM), RSL3 (3 μM), 1, 2, or 3 (0.3 μM each) for 24 h. B. Extent of OH-radical-dependent oxidation of salicylic acid (0.52 mM) by vehicle (‘w/o’, H₂O), FeSO₄ (left panel: 0.1 mM; right panel: 0.25 mM), FeCl₃ (0.25 mM), or 1 (0.1 mM) in the presence of H₂O₂ (left panel: 37 mM; right panel: 225 mM) within 30 min at room temperature. C. Non-redox-type 5-, 12-, and 15-LOX inhibitors do not interfere with cell death induction by SCs. MDA-MB-231 cells (2 × 10⁴/well of a 96-well plate) were incubated with vehicle (‘w/o’, 0.5 % DMSO), RSL3 (1 μM), 1, 2, or 3 (0.1 μM) for 48 h in the presence or the absence of Fer-1 (3 μM) or LOX inhibitors before cell viability was determined. Non-redox-type LOX inhibitors: 15-LOX-1 inhibitor BLX3887 (3 μM), 12-LOX inhibitor CAY10698 (10 μM), 5-LOX inhibitor CJ-13610 (1 μM). Redox-type LOX inhibitors: pan-LOX inhibitors baicalein (3 μM) and NDGA (3 μM). D. GPX4 protein levels of MDA-MB-231 cells (5 × 10⁵/well of a 6-well plate) incubated with vehicle (‘w/o’, 0.1 % DMSO), erastin (2 μM), RSL3 (1 μM), 1, 2, or 3 (0.1 μM each) for 24 h. E. GSH and GSSG levels of MDA-MB-231 cells (1 × 10⁶/well of a 6-well plate) incubated with vehicle (‘w/o’, 0.1 % DMSO), erastin (2 μM), RSL3 (0.3 μM), 1, 2, or 3 (0.3 μM each) for 24 h. Data are expressed as mean ± SEM from n = 3 (except n = 8 for B, left panel) independent experiments. *P < 0.05, ***P < 0.001 compared to vehicle (A, B, D, E) or the inhibitor control (C); repeated measures one-way ANOVA + Dunnett's post hoc tests (D) on log-transformed data (A, C) or ordinary one-way ANOVA + Dunnett's post hoc tests (B, left panel) or two-tailed unpaired Student t-test (B, right panel).