Fig 2: 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 3: EIF4E mediates ferroptosis independent of protein synthesis.a Western blot analysis of the indicated proteins in control and EIF4E-knockdown (EIF4EKD) HT-1080 and Calu-1 cells. b Analysis of cell viability in the indicated cells following treatment with RSL3 or erastin for 24 h. Mean ± SD, n = 3. c Analysis of cell death in the indicated cells following treatment with RSL3 (0.5 µM) or erastin (5 µM) for 24 h. Mean ± SD, n = 3. d Analysis of lipid peroxidation in the indicated cells following treatment with RSL3 (0.5 µM, 4 h) or erastin (5 µM, 6 h). Mean ± SD, n = 3. e Analysis of iron accumulation in the indicated cells following treatment with RSL3 (0.5 µM) or erastin (5 µM) for 24 h. Mean ± SD, n = 3. f Western blot analysis of the indicated proteins in HT-1080 and Calu-1 cells following treatment with RSL3 (0.5 µM) or erastin (5 µM). FBS: medium having 20% fetal bovine serum for 9 h. g, h Western blot analysis of puromycylated peptides in HT-1080 and Calu-1 cells following treatment with RSL3 (0.5 µM), erastin (5 µM), 4EGI-1 (10 µM), or 4E1RCat (10 µM). The cells were incubated with or without puromycin (10 µg/ml) for 10 min prior to cell lysis. i Western blot analysis of the indicated proteins in HT-1080 and Calu-1 cells following treatment with RSL3 (0.5 µM) or erastin (5 µM) for 9 h. j The EIF4E-knockdown HT-1080 cells (EIF4EKD1) were transfected with wild-type (WT) EIF4E or EIF4E-S209A mutant along with cap-dependent luciferase reporter cDNA. 48 h after transfection, the transfected cells were treated with 4EGI-1 (10 µM) or 4E1RCat (10 µM) and harvested for luciferase assay (RLA). Mean ± SD, n = 3; one-way ANOVA with Tukey’s multiple comparisons test; ns not significant. k Analysis of cell death in indicated HT-1080 cells following treatment with RSL3 (0.5 µM) or erastin (5 µM) for 24 h. Mean ± SD, n = 3. l Western blot analysis of protein expression in indicated HT-1080 cells.

Fig 4: The EIF4E-ALDH1B1 complex facilities ferroptosis.a HT-1080 and Calu-1 cells were treated with RSL3 (0.5 µM, 4 h). EIF4E was immunoprecipitated using an anti-EIF4E antibody and subjected to mass spectrometry analysis. b Analysis of indicated mRNA in the corresponding siRNA knockdown EIF4EKD HT-1080 and EIF4EKD Calu-1 cells (n = 3). c Cell viability of indicated cells treated with RSL3 (0.5 µM) or erastin (5 µM) for 24 h (n = 3). d Western blot analysis of protein expression in indicated cells. e Cell death analysis of indicated cells treated with RSL3 (0.5 µM), erastin (5 µM), or ferrostatin-1 (Fer-1; 1 µM) for 24 h. Mean ± SD, n = 3; two-way ANOVA with Tukey’s multiple comparisons test. f, g Immunoprecipitation (IP) analysis of EIF4E-binding proteins in indicated cells treated with RSL3 (0.5 µM), 4EGI-1 (10 µM), or 4E1RCat (10 µM) for 4 h. Mean ± SD, n = 3; one-way ANOVA with Tukey’s multiple comparisons test. IB immunoblot. h Western blot analysis of whole-cell lysates (WCL) or cell fractionation (C: cytoplasmic; M: membrane/organelle; N: nuclear/cytoskeletal) of indicated cells treated with RSL3 (0.5 µM, 4 h). i Western blot analysis of the indicated proteins in indicated cells treated with RSL3 (0.5 µM) or erastin (5 µM) for the indicated time. j, l Immunoprecipitation (IP) analysis of EIF4E-ALDH1B1 complex in whole-cell extracts (j) or membrane protein extracts (l) of Calu-1 cells treated with RSL3 (0.5 µM) and/or deferoxamine (100 µM) for 4 h. Mean ± SD, n = 3; one-way ANOVA with Tukey’s multiple comparisons test. IB immunoblot. k Analysis of intracellular iron in Calu-1 cells treated with RSL3 (0.5 µM), 20% FBS, or staurosporine (0.25 µM) for 4 h. Mean ± SD, n = 3; one-way ANOVA with Tukey’s multiple comparisons test. m His-tag affinity pull-down analysis of the binding of recombinant protein EIF4E to ALDH1B1 or ALDH3A1. n Immunoprecipitation (IP) analysis of EIF4E-ALDH1B1 complex in indicated Calu-1 cells treated with RSL3 (0.5 µM, 4 h). IB immunoblot. o Cell death analysis of indicated Calu-1 cells treated with RSL3 (0.5 µM, 24 h). Mean ± SD, n = 3.

Fig 5: EIF4E and ALDH1B1 regulates ferroptosis sensitivity in vivo.a A scheme of treatment in an HT-1080 xenograft tumor model with IKE. Athymic nude mice were injected subcutaneously with indicated ALDH1B1-knockdown (ALDH1B1KD) or EIF4E-knockdown (EIF4EKD) HT-1080 cells for 7 days and then treated with IKE (40 mg/kg, i.p., once every other day) at day 7 for 2 weeks. b Tumor volumes were calculated weekly (n = 6 mice/group; two-way ANOVA with Tukey’s multiple comparisons test; data are presented as mean ± SD). c Photographs of isolated tumors on day 14 after treatment. d–i The levels of 4HNE (d) and PTGS2 mRNA (e) in isolated tumors, serum HMGB1 (f), caspase-3 activity (g), ALDH1B1 protein (h), and EIF4E protein (i) in isolated tumors at day 14 after treatment were assayed (n = 6 mice/group; two-way ANOVA with Tukey’s multiple comparisons test; data were presented as mean ± SD).