Fig 1: A model illustrating the function of EIF4E and ALDH1B1 in regulating 4HNE-mediated ferroptosis.EIF4E promotes ferroptosis by directly blocking ALDH1B1, thereby promoting the accumulation of toxic 4HNE and subsequent NOX1-dependent lipid peroxidation.
Fig 2: 4HNE is a product and mediator of ferroptosis.a Immunofluorescence analysis showing staining of 4HNE in indicated Calu-1 cells treated with RSL3 (0.5 μM, 4 h) (scale bar = 10 µm). Mean ± SD, n = 3; two-way ANOVA with Tukey’s multiple comparisons test. b Cell viability of control and ALDH1B1-overexpressing (ALDH1B1OE) HT-1080 and Calu-1 cells treated with 4HNE for 24 h. Mean ± SD, n = 3. c Cell viability of ALDH1B1-knockdown HT-1080 and Calu-1 cells treated with 4HNE (50 μM) and/or ferrostatin-1 (Fer-1; 1 μM)/ZVAD-FMK (10 μM) for 24 h. Mean ± SD, n = 3; one-way ANOVA with Tukey’s multiple comparisons test. d Cell viability of indicated HT-1080 cells treated with RSL3 (0.5 μM) and erastin (5 μM) in the absence or presence of 4HNE (12.5 μM) for 24 h. Mean ± SD, n = 3; two-way ANOVA with Tukey’s multiple comparisons test. e, f Cell death (e, 24 h) or lipid peroxidation (f, 6 h) of indicated cells treated with 4HNE (12.5 μM), RSL3 (0.1 μM), and erastin (1 μM) in the absence or presence of ferrostatin-1 (Fer-1; 1 μM), 4EGI-1 (10 μM), 4E1RCat (10 μM), ZVAD-FMK (10 μM), necrosulfonamide (NSA; 1 µM), 2-acetylphenothiazine (2AC; 10 μM), diphenyleneiodonium (DPI; 1 μM), baicalein (10 μM), or zileuton (10 μM) (n = 3). g NOX activity of indicated cells treated with IL4 (50 ng/ml) or 4HNE (12.5 μM) plus RSL3 (0.1 μM) or erastin (1 μM) for 6 h. Mean ± SD, n = 3. h Analysis of gene expression in indicated cells treated with 4HNE (12.5 μM) in the presence of RSL3 (0.1 μM) or erastin (1 μM) for 24 h (n = 3). i Analysis of NOX1 and CYBB/NOX2 gene expression in indicated cells. Mean ± SD, n = 3. j Cell death of indicated cells treated with 4HNE (12.5 μM) plus RSL3 (0.1 μM) or erastin (1 μM) for 24 h. Mean ± SD, n = 3. k Lipid peroxidation of indicated cells treated with 4HNE (12.5 μM) plus RSL3 (0.1 μM, 4 h) or erastin (1 μM, 6 h). Mean ± SD, n = 3.
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