Fig. 1

Iron chelator deferoxamine (DFO) inhibits hinokitiol (Hino)-induced apoptosis in tri-negative breast cancer cells (TNBCs). Two TNBCs cells lines, MDA-MB-231 and 4T-1, were used in (A), one MDA-MB-231 in (B-K). A Hino effect on cell viability, detected by CCK8, in a concentration-dependent manner 24 h post-Hino treatment (8,000 cells per well). B Hino effect on cell viability within 24 and 48 h post-Hino treatment (8,000 cells per well). C The expression of iron-related proteins IRP2, TfR1, FTL, and FTH in cells treated with Hino for 24 h. D The aconitase activities of cells treated with Hino for 24 h. m-aco, mitochondrial aconitase, c-aco, cytosolic aconitase. E The activities of mitochondrial electron transport chain (ETC) complexes (activities: complex I and II) in cells treated with 100 µM Hino for 24 h. F The protein expression of mitochondrial electron transport chain (ETC) complexes (NDUFS1, SDHB, and UQCRFS1, subunits of complex I/II/III, respectively) in cells treated with Hino for 24 h. G, H Mitochondrial membrane potential detected using JC-10, and ATP content in cells treated with 100 µM Hino for 24 h. I, J The levels of apoptosis-related proteins, cleaved caspase 3, and released cytochrome C from mitochondria to cytosol, detected by immunoblotting in cells treated with 100 µM Hino for 24 h. K DFO inhibited the Hino effect on cell viability. DMSO: dimethylsulphoxide as a vehicle; Fer-1 (1 µM): ferroptosis inhibitor ferrostatin-1; Trolox (100 µM): vitamin E derivative as an antioxidant; DFO (20 µM): iron chelator deferoxamine; z-VAD-FMK (25 µM): apoptosis inhibitor; NFA (2 µM): necrosis inhibitor necrosulfonamide; VX765 (10 µM): caspase-1 inhibitor belnacasan; CA-5f (2 µM): autophagy inhibitor. *, p < 0.05; **, p < 0.01, ***, p < 0.001