Fig 2: 5-LO inhibits Pae from exerting the anti-migratory and anti-invasive effects. (A) The cell viability at 24, 48 and 72 h was estimated via Cell Counting Kit-8 assay in HeLa cells divided into control, Pae, Pae+vector and Pae+pcDNA 3.1–5-LO groups. (B and C) Colony-formation assay was performed to determine the colony-forming capacity of HeLa cells in different groups. (D and E) Wound-healing assay was conducted to evaluate the migratory capacity of HeLa cells (magnification, ×100). (F and G) Transwell assay was performed to assess the invasive capacity of HeLa cells (magnification, ×100). (H) The protein levels of MMP-2, MMP-9 in HeLa cells were detected by western blotting. **P<0.01, ***P<0.01 vs. control group. #P<0.05, ##P<0.01, ###P<0.001 vs. Pae+vector group. 5-LO, 5-lipoxygease; MMP, matrix metalloprotease.

Fig 3: Ferr-1 and 3-MA relieved iron deposition, lipid peroxidation, and ferritinophagy in IL-13-induced BEAS-2B cells. (a–c) The levels of free iron ions in these cells were detected with commercial kits. (d–f) The ratio of GSH/GSSG was determined with the commercial kits. (g) The expression of GPX4, SLC7A11, SLC3A2, and ALOX5 in these cells was determined with western blotting. (h) The expression of NCOA4, TFR1, and DMT-1 in these cells was detected with western blotting. (i) The expression of ATG5 and ATG7 in these cells was determined with western blotting. ***p < 0.001 vs. the control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. ALI+IL-13. Ferr-1: ferrostatin-1; 3-MA: 3-methyladenine; ALI: air-liquid interface; GSH: reduced glutathione; GSSG: oxidized glutathione; GPX4: glutathione peroxidase 4; SLC7A11: solute carrier family 7 member 11; SLC3A2: solute carrier family 3 member 2; ALOX5: arachidonate-5-lipoxygenase; NCOA4: nuclear receptor coactivator 4; TFR1: transferrin receptor; DMT-1: divalent metal transporter 1; ATG5: autophagy-related 5; ATG7: autophagy-related 7.

Fig 4: Inhibition of lipoxygenase-5 (5-LOX) reduced NaIO3-induced oxidative stress in ARPE-19 cells. (a, b) BODIPY C11 staining lipid peroxidation (n = 6, 35 mM NaIO3,15 h, Zileuton 75 µM). (c) FerroOrange staining for intracellular ferrous ions (75 µM Zileuton, n = 12). (d) Ferroptosis-associated protein xCT detected with western blot analysis (18 mM NaIO3 for 15 h, n = 3). (e) Oxidative stress-induced DNA damage demonstrated with 8-OHdG staining (n = 4, 35 mM NaIO3, 75 µM Zileuton, 15 h). Scale bar = 50 µm. Abbreviations: xCT: cystine-glutamate antiporter; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; Zil: Zileuton.

Fig 5: Edaravone modulates key proteins of the ferroptosis pathway in the spinal cord. (A) Schematic diagram of the experimental timeline. Wistar rats were intraperitoneally injected with 5 mg/kg edaravone daily during the first 7 days. At 2 and 7 days post SCI, tissues from spinal cord lesion epicenter were collected for western blot. At 2 days post injury the lesion epicenter and caudal spinal cord were observed from the immunofluorescence. (B,D,F,H) Representative western blot images of xCT (B), GPX4 (D), ACSL4 (F) and 5-LOX (H). (C,E,G,I) Quantifications of three western blot band of xCT (C), GPX4 (E), ACSL4 (G) and 5-LOX (I). Experiments were repeated three times. Data are expressed asmean ± standard deviation. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *p < 0.05, n = 3. (J) Diagram depicts that the ferroptosis pathway of SCI is modulated by edaravone.