Fig. 6: The effects of CH004 on cell viability and lipid ROS could be prevented by ferroptosis inhibitors.

a–c CH004-induced cell death can be prevented by ferroptosis inhibitors but not by apoptosis or necroptosis inhibitors in HepG2 cells. HepG2 cells were treated with 10 μM CH004 in the presence or the absence of the indicated concentrations of the ferroptosis inhibitor ferrostatin-1 (a), apoptosis inhibitor Z-VAD-FMK (b) or necroptosis inhibitor necrostatin-1 (c) for 24 h before analysis of the cell viability (CellTiter96® Aqueous One Solution Cell Proliferation Assay, Promega). The means at each condition are shown as percentages of DMSO (control, 100%). Means ± SDs (n = 3). d Analysis of viable cells treated with CH004 in the presence or the absence of ferroptosis inhibitor by flow cytometry. HepG2 cells were treated with CH004 in the presence or the absence of 500 μM deferoxamine or 2 μM ferrostatin-1 for 24 h, before analysis using a FITC Annexin V Apoptosis Detection Kit on an LSR Fortessa flow cytometer. Typical bright-field images for cells were recorded and are shown in Supplementary Figure 14a, and the flow cytometry charts are presented in Supplementary Figure 14b. The percentages of viable cells were defined based on the Annexin V-PI double-negative-stained cells in three independent samples. Means ± SDs (n = 3). e–g CH004 increases lipid ROS in a dose-dependent manner, which can be counteracted by deferoxamine. After treatment with indicated concentrations of CH004 in the absence (e) or presence (f) of the ferroptosis inhibitor deferoxamine for 24 h, HepG2 cells were stained with 2 μM BODIPY^® 581/591 C11, a lipid ROS tracker, before the analysis with flow cytometry (Materials and Methods). The FACS histogram plot for green fluorescent (530 nm) was shown in e or f, and the quantitative data was normalized with the control (DMSO, 100%) and shown in g. The ratio between green fluorescence (oxidized BODIPY^® 581/591 C11) and red fluorescence (reduced BODIPY^® 581/591 C11) was additionally displayed (e, f). Means ± SDs (n = 3). h CH004 dose-dependently depletes the intracellular GSH in HepG2 cells. HepG2 cells were seeded in a 6-well culture plate for 24 h before the treatment of CH004 at the indicated concentration for 24 h. Then, the cells from two wells were lysed with 100 μl, 50 mM MES buffer containing 1 mM EDTA (pH 6–7) before the deproteination, and the intracellular total GSH was measured with a commercial glutathione assay kit (Cayman, see “Materials and Methods” section for detailed protocol). The amount of total GSH was normalized with the corresponding protein concentration (BCA protein assay reagent kit, Pierce) and displayed as a percentage of control (DMSO, 100%). Means ± SDs (n = 3). Statistical analyses were performed on the raw data by one-way ANOVA with Bonferroni post-tests. ***p < 0.001. All the experiments were independently repeated twice and one representative result is present; n.s. no significance