Fig. 3. Detection of G₁-phase arrest in Ras-NIH3T3/Mdr cells treated with rapamycin. (A) Pro-apoptotic activity of caspase-3 was determined by detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA in rapamycin- or paclitaxel-treated (positive control) cells. Values represent the mean ± SD of duplicate determinants from one of three representative experiments. **P < 0.01 as determined by the Dunnett’s T-test. (B) Cell cycle progression was assessed by staining fixed cells with propidium iodide to estimate the percentage of cells in the G₁ (2N DNA content), G₂/M (4N DNA content), and S phases (2 to 4N DNA content). The percentage of cells in each phase of the cell cycle was quantitated using Cell-Quest Pro software and plotted. (C) Whole cell extracts were prepared 48 h post-rapamycin treatment. The expression of p21^Cip1 and p27^Kip1 were assessed by immuno-blotting. β-actin was assessed as a loading control. The results presented are representative of at least three independent experiments.