FIG. 6.

Inhibition of Mnk-dependent eIF4E phosphorylation by a Mnk1 inhibitor (C) augments rapamycin-mediated growth inhibition (A and B) in human lung cancer cells. (A) The individual cell lines, as indicated, were seeded in 96-well plates. On the next day, they were treated with the indicated concentrations of rapamycin (Rap) alone, 2.5 μM CGP57380 (CGP) alone, and their combination. After 3 days, the plates were subjected to determination of cell numbers using the sulforhodamine B assay. The bars are means of four replicate determinations plus standard deviations. (B) H460 cells at a density of ∼ 250 cells per well were seeded in 12-well plates. On the second day, the cells were treated with the indicated concentrations of rapamycin (Rap) alone, CGP57380 (CGP) alone, and their combination. The same treatments were repeated every 3 days. After 10 days, the plates were stained for the formation of cell colonies with crystal violet. The colonies in each well were counted. The bars are means of three replicate determinations plus standard deviations. (C) H157 cells were pretreated with the indicated concentrations of CGP57380 (CGP) for 30 min and then cotreated with 10 nM rapamycin (Rap) for 1 h or 24 h. The cells were then subjected to preparation of whole-cell protein lysates for detection of the indicated proteins using Western blotting.