Fig. 1: GPX4 inhibition can trigger ferroptotic and apoptotic cell death.

A, B Quantification of cell death, calculated as percentage of PI-positive cells. Cells were stimulated with 4.5 µM RSL3 (HT1080_S), 300 nM RSL3 (HT1080_S), 50 µM QVD and 2 µM ferrostatin-1 (FER). Data are the mean ± range of technical duplicates (A) or mean ± SEM of technical triplicates (B). Panels show one representative examples of at least three independent experiments. C Cell death was determined for cells cultured in presence or absence of 50 µM QVD and decreasing amounts of FER. Data are means ± SEM of technical triplicates of one representative example of three independent experiments. D Cells were stimulated with 4.5 µM RSL3 for 24 h and analysed for lipid peroxidation (C11 BODIPY 581/591 conversion) via flow cytometry. Data show mean values ± SD of three independent experiments. Asterisks indicate statistical significance compared to the control group (*p ≤ 0.05, **p ≤ 0.01; One-way Anova with Tukey’s post hoc test). E Cells were stimulated with 2.25 µM RSL3 and monitored at 10 min intervals via life-cell-imaging. Data are the mean ± SD of three independent experiments. F Data show median and quartiles of cells pooled from three independent experiments. Asterisks indicate statistical significance (***p ≤ 0.001; Mann-Whitney test). G Subcellular analysis of cytochrome-c redistribution. Cells were stimulated with 2.25 µM RSL3 for the indicated time points or with 10 µM ABT-199 + 10 µM S63845 + 50 µM QVD for 4 h as apoptotic positive control (PC). Fixed cells were immunostained for cytochrome-c and segmented based on MitoTracker signals. Scale bar in representative images, 20 µm. Data show means and scatter from one out of three representative experiments, with about 100 cells per condition. Asterisks indicate statistical significance compared to the control group (****p ≤ 0.0001; ns = not significant, One-way Anova with Tukey’s post hoc test). H Cells were stimulated with 4.5 µM RSL3 or 10 µM ABT-199 + 10 µM S63845 + 50 µM QVD (Positive ctrl. = PC) and fractioned into cytoplasm and pellet containing the mitochondria. One representative of three independent experiments is shown. I Immunoblotting of caspase-3 (C3) and PARP cleavage in HT1080_S cells upon RSL3 (4.5 µM) treatment for 16 h, one representative of three independent experiments is shown. J Immunoblots of BAX, BAK and GPX4 expression in vector control cells (VC) and HT1080_S (Bax/Bak)^−/− clones. K Quantification of cell death, calculated as percentage of PI-positive cells. Cells were stimulated with 2.25 µM RSL3, 50 µM QVD and 2 µM FER. Data show the mean ± range of technical duplicates of one representative example of four independent repeats.