Fig 2: Inhibition of ferroptosis by Fer-1 alleviated oxidative stress-induced brain injury via upregulating the expression of GPX4. (a) Western blotting assay for XCT, GPX4, CoQ10B, and FSP1 expression in different groups in vivo. (b–e) Quantification of the expression of XCT, GPX4, CoQ10B, and FSP1. (f) Quantitative analysis of MDA in different groups in vivo. (g) Western blotting assay for the expression of GPX4 in all groups with different doses. (h) Quantification of GPX4 expression. (i) Western blotting assay for the expression of XCT, GPX4, CoQ10B, and FSP1 in different groups in vitro. (j–m) Quantification of the expression of XCT, GPX4, CoQ10B, and FSP1. (n) Quantitative analysis of MDA. n = 6/group. Bars represent the mean ± SEM. *P < 0.05 and **P < 0.01. GPX4: glutathione peroxidase 4; FSP1: ferroptosis suppressor protein 1; MDA: malondialdehyde.

Fig 3: Chronological changes in ferroptosis-associated genes and oxidative stress levels in h9c2 cells after rapid pacing. (a–d) Chronological changes of FTH1, GPX4, SLC7A11, and FTL miRNAs after rapid pacing. (e, f) Representative gel bands depicting FTH1, GPX4, and SLC7A11 proteins expression in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (g, h) Representative gel bands depicting FTH1, GPX4, and SLC7A11 proteins expression in h9c2 cells stimulated by rapid pacing for 72 h and treated with or without Fer-1. (i, k) Representative images of immunofluorescence staining for FTH1 proteins in h9c2 cells stimulated by rapid pacing for 48 h with or without Fer-1. (j, l) Representative images of reactive oxygen species by fluoroscopy stimulated by rapid pacing for 48 h with or without Fer-1. (m, n) Mitochondrial membrane potential was detected through flow cytometry in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (o) Total iron level in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (p) MDA level in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. (q, r) Representative gel bands depicting ion channel expression in h9c2 cells stimulated by rapid pacing for 48 h and treated with or without Fer-1. Data are presented as the mean ± SD, n =3. Statistical significance was determined using Student's t test (a–d) or one-way ANOVA with a post hoc Dunnett test (e–r). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. Pacing group.

Fig 4: Knockdown of SLC7A11 get susceptibility to erastin-induced ferroptosis in mature MO3.13 cells. (A) RT–qPCR analysis of the expression of ferroptosis marker SLC7A11 in mature MO3.13 cells. Gene expression levels were normalized to those of RPL27; three biologically independent samples (n = 3) per group. Data are presented as means ± SEM. **P < 0.01 and ***P < 0.001 by Student's t-test. (B) Cell viability was measured using a WST-8 assay; three biologically independent samples (n = 3) per group. Data are presented as means ± SEM. ***P < 0.001. One-way ANOVA followed by Tukey’s post hoc test.

Fig 5: DMF reduces ferroptosis in IRI. (a) KEGG pathway classification and enrichment of differentially expressed genes in the sham versus IR groups. (b) Representative transmission electron microscopy images of mitochondria in hepatocytes of the liver. Red arrow: damaged mitochondria. (c) the level of lipid peroxide malondialdehyde (MDA) in each group. (d) the relative GSH/GSSG ratio in the liver of mice. (e) Representative immunohistochemistry (IHC) staining of NRF2 in the liver of each group, and NRF2 positive areas were marked with arrows. (n = 6, original magnification, ×20, Scale bar, 50 µm). (f) Western blot and quantification of hepatic GPX4, COX2, NRF2, SLC7A11, and HO-1 proteins. (g) Relative mRNA expression of GPX4, COX2, NRF2, SLC7A11, and HO-1 in each group. The results are shown as mean ± SD of six mice in each group. *** P <0.001, ** P <0.01, * P <0.05 (analyzed by one-way analysis of variance, ANOVA).