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. 2024 Jun 26;75:103257. doi: 10.1016/j.redox.2024.103257

Fig. 2.

Fig. 2

SCs trigger excessive phospholipid peroxidation in triple negative breast cancer cells. A, B. MDA-MB-231 cells (2 × 104/well of a 96-well plate) were treated with vehicle (‘w/o’, 0.5 % DMSO), 1, 2, 3 (A: 1 μM each; B: 0.1 μM each) or RSL3 (1 μM) in the presence or the absence of cell death inhibitors for 48 h before cell viability was determined. A. Phase contrast microscopic images (scale bar: 500 μm). B. Inhibition of cell death induction by ferroptosis inhibitors but not by other cell death inhibitors. Ferroptosis inhibitors: Fer-1 (3 μM), ciclopirox (0.25 μM), N-acetyl-l-cysteine (NAC, 2.5 mM), β-mercaptoethanol (β-ME, 200 μM), necrostatin-1 (Nec-1, 40 μM; also inhibits necroptosis); necroptosis inhibitor: necrostatin-2 (Nec-2, 10 μM), apoptosis inhibitor: Q-VD-OPh (20 μM); autophagy inhibitors: wortmannin (1 μM), 3-methyladenine (3-MA, 1 mM); pyroptosis inhibitor: MCC950.Na (1 μM). C. Cellular ROS formation of MDA-MB-231 cells (2 × 104/well of a 96-well plate) treated with vehicle (‘w/o’, 0.5 %), RSL3 (1 μM), 1, 2, 3 (1 μM each), or H2O2 (1 mM) for 2 h. D, E. Flow cytometric analysis of liperfluo-stained lipid peroxides in MDA-MB-231 cells (5 × 105/well of a 6-well plate) treated with vehicle (‘w/o’, 0.5 % DMSO), RSL3 (1 μM), 1 (1 μM), or 1 together with Fer-1 (3 μM) for 6 h. D. Histogram showing cell number as a function of liperfluo staining. E. Quantitative analysis of liperfluo-positive and -negative cells based on the data from panel D. F–I. MDA-MB-231 cells (3.12 × 106/75 cm2) were treated with vehicle (‘w/o’, 0.1 % DMSO), 1, 2, 3 (1 μM each, unless otherwise stated), or RSL3 (1 μM) for 2 h, and oxidized and non-oxidized PE, PC, and PI species were analyzed by UPLC-MS/MS. F. Volcano plot showing the log2 of fold-change in the amount of (per)oxidized PC, PE, and PI species relative to vehicle control and the negative log10 (adjusted P value) calculated vs. vehicle control; two-tailed multiple unpaired Student t tests with correction for multiple comparisons (false discovery rate 5 %). G. Amount of PE(18:0_20:4 + 3[O]). H. Extracted chromatograms based on the fragmentation of [PE(18:0_20:4 + 3[O]-H]- to [20:4 + 3[O]-H]-. I. Percentage changes in the proportion of SFA-, MUFA-, and PUFA-containing PC and PE. Data are expressed as mean ± SEM from n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to vehicle control or indicated by bars; repeated measures one-way + Dunnett's tests on log-transformed data (G) or repeated measures two-way ANOVA + Dunnett's (I) or Tukey's post hoc test (E) or two-tailed unpaired Student t-test (C, G, RSL3).