Figure 7. Altered phosphorylation of eIF4B on Ser422 is responsible for synergic inhibition of cell survival by combined blockage of Pim and PI3K/Akt/mTOR pathways.

A. interference efficiency of shRNA-based knockdown of eIF4B in K562 cells was determined by Western blotting. shRNA targeting luciferase (sh-luc) served as a control. B. protein levels of eIF4B in A were quantitated by densitometry and normalized to β-actin. Plotted are the average levels from three independent experiments. The error bars represent the SEM (**P < 0.01). C. K562 cells in A were treated with SMI-4a alone, rapamycin alone, or their combination at indicated concentrations. After 36 h, the cells were assessed for apoptotic fraction. Values represent means ± SEM, n = 3 (*P < 0.05, **P < 0.01). D. K562 cells ectopically expressing empty vector, eIF4B-WT or eIF4B-S422E mutant were examined by Western blotting. E. cells in D were treated with 12.5 μM SMI-4a alone, 10 μM Akti-1/2 alone, or their combination. After 48 h, cells were harvested for Annexin V staining as described in C. F. cells in D were treated with 12.5 μM SMI-4a alone, 5 μM rapamycin alone, or their combination for 48 h. Apoptosis was examined as described in C. G. K562 cells expressing vector, eIF4B-WT or eIF4B-S422E mutant were transplanted into nude mice. At the 11^th day after inoculation, mice bearing xenografts were treated with mock or combination of 50 mg/kg SMI-4a and 10 mg/kg rapamycin each other day. At the 29^th day mice were sacrificed. Shown are representative tumors under mock or drug administration. H. the relative tumor volumes were displayed as described in Figure 6B. The average volume of tumor (empty vector) with mock treatment was set as 100%. Error bars, SEM, n = 18 (*P < 0.05). I. tumors in G were immunoblotted with indicated antibodies. Shown are representative results from three independent experiments.