Fig. 5

2DG perturbs two global protein synthesis pathways. a In the bicistronic reporter, the cap-dependent 5′UTR of cytomegalovirus (CMV) drives synthesis of renilla luciferase, whereas the IRES of hepatitis C virus (HCV) allows cap-independent translation of firefly luciferase [35]. b NALM-6 cells, immediately after electroporation with the dual reporter in a (see Methods), were incubated with 0, 1, 5, or 10 mM 2DG for 6 h, or treated for 6 h with silvestrol (100 nM) to inhibit cap-dependent initiation of translation or cycloheximide (CHX, 20μg/mL) to inhibit polypeptide elongation. Luminescence from renilla or firefly luciferase (see Methods) is plotted as mean ± SD (n = 3). Significance was determined by one-way ANOVA: ****P < 0.0001. Renilla luciferase clearly reflected cap-dependent initiation, because silvestrol, which inhibits initiation by interfering with RNA helicase eIF4A [21], abolished renilla but not firefly luciferase, whereas elongation-inhibitor cycloheximide markedly reduced both. The elevation in firefly luciferase by silvestrol is consistent with considerable evidence that marked suppression of cap-dependent translation boosts IRES-driven translation [34]. c Impact of 2DG on complexes with cap-binder eIF4E, analyzed by pull-down on m7GTP-sepharose beads, which bind eIF4E. Protein extracts from NALM-6 cells treated 6 h with 1 or 10 mM 2DG, with or without 10 mM mannose, were incubated with m7GTP-sepharose beads. Bound proteins were denatured and subjected to western blotting to reveal cap-associated eIF4E bound to eIF4G (active complexes) or to 4E-BP1 (inactive complexes). A representative blot is shown (n = 3). d The ratio of eIF4G/4E-BP-1 bound to eIF4E was quantified from three independent experiments like that in c, analyzed with ImageJ software. Data are plotted as mean ± SD. Significance of the difference from the untreated control (lane 7) determined by one-way ANOVA: *P < 0.05, **P < 0.01, or ns. e Western blot analysis of several pathways that influence the initiation and elongation stages of translation. The proteins analyzed here and in d affect formation of complexes critical for cap-dependent initiation (p70S6K, S6, eIF4E, eIF4G, 4E-BP1) or control of elongation (eEF2K and eEF2). f The levels of some short-lived proteins are affected more than others by the capacity of 2DG to inhibit global protein synthesis. Western blot analysis reveals that 2DG downregulates the short-lived oncoproteins MDM2 and c-MYC, but not cyclin D1. Supplementary Fig. 6 shows others unaffected by 2DG