Fig. 2. EIF4E mediates ferroptosis independent of protein synthesis.

a Western blot analysis of the indicated proteins in control and EIF4E-knockdown (EIF4E^KD) 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 (EIF4E^KD1) 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.