Fig. 3: The EIF4E-ALDH1B1 complex facilities ferroptosis.
From: A noncanonical function of EIF4E limits ALDH1B1 activity and increases susceptibility to 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.
