Fig. 2. 4EBP1/2 maintain antioxidant power and preserve the redox balance under glucose starvation.

A Control (shScr) and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose starved (Glc strv) for the indicated times and labeled with azidohomoalanine (AHA). Levels of AHA-labelled proteins were detected by immunoblotting. B WT and 4E KO MEF were grown in complete medium or glucose starved (Glc strv) for the indicated times, labeled with EdU and analyzed by flow cytometry. C WT and 4E KO MEF were grown in complete medium or glucose (Glc) starved for 24 h. Mitochondrial membrane potential (Δψm) with TMRE staining and mitochondrial mass were measured. D Control (shScr) and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 24 h and the corresponding metabolites were measured by LC-MS. E NAD⁺ and NADH levels measured in (C) were plotted as NADH/NAD⁺ ratio. F NADP⁺ and NADPH levels measured in (C) were plotted as NADPH/NADP⁺ ratio. G WT and 4E KO MEF were grown in complete medium or glucose (Glc) starved for 24 h, and NADP⁺ and NADPH levels were measured. H Scheme of the usage of NADPH in recycling oxidized glutathione for H₂O₂ detoxification. (I) WT and 4E KO MEF were grown as in (C), and reduced and total glutathione were measured and expressed as the ratio of reduced (GSH) to oxidized (GSSG) glutathione. J WT and 4E KO MEF were grown as in (C), and total glutathione was measured. K WT and 4E KO MEF grown as in (C) were labelled with CM-DCFDA and analyzed by flow cytometry. L 4E KO MEF were grown in glucose starved medium (Glc strv) and treated with vehicle (V), N-acetyl cysteine (NAC) or Catalase (CAT) for 48 h. Cell death was measured by PI staining and flow cytometry. Data are shown as the mean ± SD. Statistics: unpaired one-sided Student’s t test (A–C, E–G, I–L); n = 3 independent experiments for (A–C, E–G, I–L). Source data are provided as a Source Data file.