Figure 5. Simultaneous Mnk inhibition increases rapamycin-mediated inhibition of cell proliferation and colony formation.

(A) Daoy cells were incubated for five days with increasing concentrations of CGP57380 (1, 5, 10, 50 μM) in the presence or absence of increasing concentrations of rapamycin (1, 5, 10, 50 nM, upper panel) or OSI-027 (1, 5, 10, 50 μM, lower panel). Subsequently, cells were subjected to WST-1 proliferation assays. Means ± SE of the values from 3 independent experiments (each done in triplicates), are shown. Data are expressed as percentages of control DMSO treated samples. (B) Daoy cells were treated with the indicated concentrations of CGP57380, in the presence or absence of the indicated concentrations of rapamycin or OSI-027. After five days, cell numbers were counted using an automated cell counter. Means ± SE are shown as values of 3 independent experiments. Data are expressed as percentages of control DMSO treated samples. (C) Daoy cells were plated in soft-agar and treated with CGP57380 (10 μM) with or without rapamycin (10 nM) or OSI-027 (0.5 μM). After 7 days, colony formation was quantified using the fluorescent cell stain CyQUANT GR Dye (Cell Biolabs Inc.) in the Synergy HT Plate reader. Means ± SE of the values from 4 independent experiments are shown. Data are expressed as percentages of DMSO treated samples. *, p < 0.05, **, p < 0.005, ***, p <0.0005 using a paired t-test (D) Daoy cells were transfected with control, Mnk1, or Mnk2 siRNA. After 48 hours, cells were counted and equal numbers were plated in soft-agar and treated with DMSO as a control, rapamycin (10 nM) or OSI-027 (0.5 μM). Cells were incubated for 7 days. Colony formation was quantified using the fluorescent cell stain CyQUANT GR Dye (Cell Biolabs Inc.) in the Synergy HT Plate reader. Means ± SE are shown from 6 independent experiments. Data are expressed as percentages of DMSO treated control siRNA transfected samples. *, p < 0.05 using a paired t-test.