S6 RP dephosphorylation in G0/G1-gated cells occurs prior to substantial cell cycle arrest. Cells were exposed to GSI (DAPT) or vehicle (DMSO) for the indicated amount of time. At each time point, cells were intracellularly stained with an antibody specific for phospho-S6 RP and their DNA contents measured by propidium iodide staining. Cells in the G₀/G₁ fraction were gated based on DNA content and their level of S6 RP phosphorylation was determined. The fraction of the mean fluorescent intensity (MFI) of GSI-treated cells to the MFI of mock-treated cells was calculated and plotted over time (open circles, left Y-axis). The ratio of the percentage of cells in G₀/G₁ phase in GSI-treated populations to that in mock-treated populations was measured over 8 days to monitor cell cycle arrest (solid circles, right Y-axis; axis is reversed to facilitate comparison between the 2 graphs). Data shown are representative of 3 independent experiments with similar results.