Proliferation of cultured tumor cells at various concentrations of Cdk4 inhibitor. HeLa/Fucci2 (A, B, and C) or NMuMG/Fucci2 (D, E, and F) cells were plated at the same density in multiple wells and grown in the presence of various concentrations of Cdk4 inhibitor. Following 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 draw cell proliferation curves (A and D). In addition, the number of red-, orange-, and yellowish green-emitting cells were determined and the averaged fluorescence intensities of individual nuclei were counted. The cell cycle profiling data with various concentrations of the compound at 12 and 24 h are shown (B and E). Data points are means ± SD of triplicate samples (wells). Two representative fluorescence images of HeLa/Fucci2 (C) and NMuMG/Fucci2 (F) cells that were treated with 3 μM Cdk4 inhibitor for 24 h are shown. Scale bar, 10 μm.