Proliferation of cultured tumor cells at various concentrations of etoposide. HeLa/Fucci2 (A and B) or NMuMG/Fucci2 (C and D) cells were plated at the same density in multiple wells and grown in the presence of the indicated concentrations of etoposide. Following a culture period of 0, 12, 24, 36, or 48 h, cells were fixed, and total cell numbers were counted after nuclear staining with DAPI to generate cell proliferation curves (A and C). Additionally, the number of red-, orange-, and yellowish green-emitting cell populations as well as the average fluorescence intensities of individual nuclei were automatically counted. The cell cycle profiling data with various concentrations of the compound at 12 and 24 h are shown with the Fucci colors (B and D). Because Fucci probes utilize protein degradation mechanisms and reflect accumulation of Cdt1 and Geminin, their signal intensities (the average fluorescence intensities of individual nuclei) depend on the duration of cell-cycle phases. The prolongation of the G2 phase in the presence of 1 μM etoposide is indicated by black arrows. Data points are means ± SD of triplicate samples (wells).