4EGI-1 but not RAD001 reduces the translation rate of c-Myc mRNA in the MOLM-14 AML cell line. Cells from the MOLM-14 leukemic cell line were cultured 12 hours at 106/mL in αMEM containing 10% FCS, without or with 10 nM RAD001, 25 μM LY294002, or 100 μM 4EGI-1, and then cell lysates from 108 cells were subjected to sucrose density centrifugation and processed as described in “Polysome analysis.” Gradients were fractioned using a Beckman fraction recovery system connected to a LKB UV detector. UV recording is shown in the top level of the figure. Twelve fractions were collected, and ribosome content of each fraction was determined by Agilent 2100 electrophoresis for both 18S and 28S rRNA (A, bottom panel). RNA was extracted in each fraction separately and quantitative reverse-transcribed PCR for c-Myc expression was performed; results are expressed together with rRNA quantifications results in the lower panel of the panel A. From these experiments, we determined that free ribosome subunits sedimented in fractions 4 and 5. Accordingly, fractions 6 to 12 were considered to contain polysomes. The nonpolysome (fractions 1-5– and polysome (fractions 6-12)–containing fractions were then pooled and analyzed by real-time quantitative PCR for c-Myc mRNAs. Results are expressed as a percentage of nonpolysomal-bound or polysomal-bound c-Myc mRNA relative to the total c-Myc mRNA amount (B).
