Fig. 2: Inhibition of ferroptosis by ferrostatin-1, upon deprivation of antioxidants, facilitates neuronal differentiation. | Nature Communications

Fig. 2: Inhibition of ferroptosis by ferrostatin-1, upon deprivation of antioxidants, facilitates neuronal differentiation.

From: Suppression of ferroptosis by vitamin A or radical-trapping antioxidants is essential for neuronal development

Fig. 2

a Scheme of cortical neuronal differentiation using three conditions (i) standard media with antioxidant-containing B27 (+ AO), (ii) media with antioxidant-deficient B27 containing vitamin A (-AO + vAlo), or (iii) media with antioxidant-deficient B27 containing vitamin A, which was further supplemented with ferrostatin-1 (Fer-1) (-AO + vAlo + Fer-1). b C11-BODIPY staining of day 20 immature cortical neurons generated with antioxidant (+ AO) or without antioxidants (-AO + vAlo) or supplemented with ferrostatin-1 (-AO + vAlo + Fer1) using flow cytometry. (Left) flow cytometry histograms; (right) median intensity. Data are mean ± SD of n = 3 biologically independent replicates; one-way ANOVA with Tukey’s test. c (Left), MAP2 immunofluorescence staining of cortical neurons at day 40 generated with antioxidant (+ AO) or without antioxidants (-AO + vAlo) or supplemented with ferrostatin-1 (-AO + vAlo + Fer-1). (Right), high-content image analyses. n = 3 biologically independent replicates (with approx. 30–70 segments for individual samples); Kruskal-Wallis with Dunn’s test. Scale bars, 100 µm. d MAP2 mRNA expression of cortical neurons generated with antioxidant (+ AO) or without antioxidants (-AO + vAlo) or supplemented with ferrostatin-1 (-AO + vAlo + Fer-1) using quantitative RT-PCR. Data are mean ± SD of n = 3 biologically independent replicates; one-way ANOVA with Tukey’s test. e Area of organoids at day 60 generated with antioxidant (+ AO) or without antioxidants (-AO + vAlo) or supplemented with ferrostatin-1 (-AO + vAlo + Fer-1) and measured by ImageJ. Data are mean ± SD of n = 5 independent organoids; one-way ANOVA with Tukey’s test. f (Left), organoid sections stained for the ferroptosis marker TfR1 at day 60 of forebrain generation. (Right), measurement of the TfR1 fluorescence intensity using ImageJ. Data are mean ± SD of n = 3 biologically independent replicates; one-way-ANOVA with Tukey’s test. Scale bars, 50 µm. g (Left), organoid sections stained for 4-HNE lipid peroxidation marker at day 60 of forebrain generation. (Right), measurement of the 4-HNE intensity using ImageJ. Data are mean ± SD of n = 3 biologically independent replicates with 5 image sections per replicate; one-way-ANOVA with Tukey’s test. Scale bars, 50 µm. Source data are provided as a Source Data file.

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