EIF4F complex formation is mTORC1-independent in AML and is totally abrogated by the 4E-BP1 mimetic 4EGI-1. (A) Blast cells from 6 AML patients and from the MOLM-14 leukemic cell line were cultured for 24 hours in αMEM containing 10% FCS without or with 10 nM RAD001, 25 μM LY294002, or 50 or 100 μM 4EGI-1, and then lysed. Supernatants were clarified, m⁷-GTP affinity assay was performed, and beads were solubilized in boiling Laemmli sample buffer. Western blots were performed with anti-eIF4G, anti-eIF4E, and anti–4E-BP1 antibodies. (B) Quantifications of the signal intensity of Western blots from the 6 m⁷-GTP affinity assay experiments performed in primary AML samples were done using the Multi Gauge, Version 3.0 software from Fuji. Each histogram represents the mean of 6 independent experiments, and results are expressed as the ratio between 4E-BP1 and eIF4E and between eIF4G and eIF4E, relative to the control condition (C; extracts from cells incubated without inhibitor). Vertical bars represent SD.