Figure 2

EM011 perturbs cell-cycle progression of A549 lung cancer cells. Cells were harvested for analysis at the noted times, fixed and stained with propidium iodide, and analyzed by flow-cytometry using the Cell Quest Software. A. Effect of 25 μM EM011 on cell-cycle progression of A549 cells over time shown in a three-dimensional disposition. X-axis, intensity of propidium iodide fluorescence, is indicative of the total DNA content of cells in various phases of the cell-cycle. Y-axis, number of cells detected for a given DNA content. Z-axis, time points (i.e., 0, 6, 12, 18, 24, 36, and 48 hrs). Representative results of three independent experiments. B. Quantitative graphical representation of the percentage of G2/M and sub-G1 cell population. Points, average of three independent experiments; bars, SD (p < 0.05). C. A bar-graphical representation of relative percentage of cells in various cell-cycle phases over time of drug treatment. A549 cells achieved a maximal G2/M population at 12 hrs (shown in green). D. Representative dot-plots depicting mitotic population at 0 and 12 hrs of drug-treatment as quantitated by MPM-2 and PI staining, flow-cytometrically. MPM-2, a well known phospho-serine/threonine antibody is recognized for its versatility because it can detect several mitotic phospho-proteins that are phosphorylated either directly or indirectly by M-phase-promoting factor (MPF) upon entry into mitosis. E is a bar-graphical representation highlighting the near inverse-correlation between the mitotic and sub-G1 population of cells in A549 cells. The values and error bars shown in the graph represent the averages and standard deviation (SD), respectively, of three independent experiments; (p < 0.05).