Fig. 2

FK866 inhibits the signaling cascades controlling protein synthesis in T-ALL cell lines. a RNA synthesis was determined by monitoring EU incorporation with Click-it chemistry. Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 5 μM Actinomycin D, an RNA synthesis blocking agent. The histogram quantifies the dose-dependent transcription inhibition induced by FK866 in the viable cell population. In the lower part, Click-it chemistry based on the incorporation of an aminoacid analog (AHA) was used to monitor protein synthesis. Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 350 μM Cycloheximide, as a positive control for protein synthesis inhibition. The histogram quantifies FK866-induced protein synthesis arrest in the viable cell population. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 50000 events/sample. b Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866. Thereafter, cells were lysed and the levels of total and p-Akt (Ser-473), total and p-MTOR (Ser-2448), total and p-4EBP1 (Ser-65 and Thr-70), c) total and p-EIF4E (Ser-209), total and p-EIF2A (Ser-51) were detected by immunoblotting. d Molt-4 cells were treated with FK866 for 48 h and the levels of total 4EBP1 and p-4EBP1 were evaluated. e Western blot analysis as in d in SupT1 cells. b-e, one representative experiment out of at least three biological replicates is presented and β-actin was used as loading control