Figure 5. 4E-BP1 Mediates the Effects of AKT and MEK Activation on Cap-Dependent Translation and Survival.

(A) HCT116 cells were treated with 50 nM PD0325901 and 1 µM AKTi, alone or in combination for 6 h. Cell lysates were precipitated with m⁷GTP sepharose beads followed by immunoblotting with the indicated antibodies.

(B) siRNAs against the indicated genes or control siRNAs were transfected into HCT116 cells and incubated for 72 h. Cell lysates were immunoblotted with the indicated antibodies.

(C) siRNAs against the indicated genes or control siRNAs were transfected into HCT116 cells and incubated for 30 h. The cells were then transfected with a bicistronic luciferase reporter plasmid for 24 h, and then treated with 50 nM PD0325901 and 1 µM AKTi, alone or in combination for an additional 12 h. The inhibition of cap-dependent translation was determined as in Figure 3C. Values represent means ± SEM (n=3).

(D) Detection of eIF4E and β-actin by immunoblot in HCT116 cells expressing the indicated transgenes.

(E) HCT116 cells expressing eIF4E or control vector were transfected with a bicistronic luciferase reporter plasmid for 24 h, and then treated with 50 nM PD0325901 and 1 µM AKTi, alone or in combination for an additional 12 h. The inhibition of cap-dependent translation was determined as in Figure 3C. Values represent means ± SEM (n=3).

(F) siRNAs against 4E-BP1 or control siRNAs were transfected into HCT116 cells and incubated for 48 h. The cells were then treated with 50 nM PD0325901 and 1 µM AKTi, alone or in combination for the indicated additional times. Apoptosis was assessed by sub-G1 fraction of the cells. The results are expressed as the increased levels of apoptosis by subtracting each of the DMSO-treated controls and presented as means ± SEM (n=3).