Fig. 3: Ferroptosis inhibitors ameliorates the neuronal death induced by hypoxia in vitro.

A Primary neurons were exposed to 1% O₂ for 3, 6, and 9 days. The cell viability was accessed by CCK8 assays (n = 3). B The content of intracellular ferrous ions exposed to hypoxia for 9 days was detected by FerroOrange probe. Scale bar is 20 μm. C The iron levels are determined based on the intensities measured by ImageJ (n = 6). D The GSH content and E the GSH/GSSG ratio in the primary neurons under hypoxia exposure. F The cell viability exposed by hypoxia with or without Fer-1 (1 μmol/L) administration (n = 3). G Quantitative analysis of the number of double-positive cells as a proportion of total cells (n = 6). H PI staining was utilized in primary neurons viability assessment (red for PI, blue for DAPI), PI/DAPI dual-positive cells were considered to dead cells. Scale bar is 200 μm. I Cell death was measured by flow cytometry labeled PI staining. J The levels of C11-BODIPY in each group were determined by FACS analysis. K Quantitative analysis for primary neurons lipid peroxidation (the ratio of green fluorescence to red fluorescence) under hypoxia or Fer-1 administration (n = 6). L The representative pictures of lipid peroxidation assay (red for reduction, green for oxidation). Scale bar is 50 μm. Data were showed as mean ± standard error of mean (SEM) of at least three independent experiments. Statistical analyses were carried out using two-way ANOVA and multiple t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Source data are provided as a Supplemental Material file.