Effects of ZSTK474 in combination with Rapamycin on cells. Cells were treated with the indicated doses of ZSTK4747, Rapamycin, the combination of the former two inhibitors or vehicle control for 3 days. Additive, synergistic, or antagonistic inhibitory effects of the drug combination on cell viability were determined when the experiment values (cell viability percentages) of the drug combination were overlapped with, lower than, or higher than Bliss theoretical values respectively (A). Western blot analysis was performed to determine the inhibitory effects of ZSTK474 in combination with Rapamycin on class I PI3K activity (B). Cells were seeded at a density of 20,000 cells per ml overnight, followed by treatment with 5 μM ZSTK474 (10 μM ZSTK474 in REM cells), 100 nM Rapamycin, the combination of the former two inhibitors or vehicle control for 18 hrs. Whole cell lysates (comprising 50 μg total protein) were subjected to western blot with the indicated antibodies. β-actin was used as a loading control.