Figure 1: Fucci introduction into the murine melanoma cell line B16BL6.

(a) Cell-cycle analysis of Fucci-expressing B16BL6 murine melanoma cells by flow cytometry. First, the cells were divided into three subpopulations: mAG-hGeminin-negative, mAG-hGeminin-positive, and mKO2-Cdt1-mAG-hGeminin double-positive. The DNA content of each population was analysed by staining with Hoechst 33342 dye (histograms). The coloured lines in the right-hand panels represent histograms of the cells shown in the left-hand panels. These data confirm that mAG-negative cells (i.e. G0/G1 cells) have 2N-DNA content, while mKO2-positive and mAG-positive cells (i.e. early S cells) have greater DNA content, and mKO2-negative and mAG-positive cells (i.e. late S/G2/M cells) even greater DNA content. The mAG-hGeminin-negative population was further divided into mKO2-Cdt1-negative, -moderately positive, and -highly positive cells (i.e. the early-, late-, and retained-G0/G1 phases, respectively). (b) Representative images of cell-cycle changes in Fucci-expressing B16BL6 cells visualised by time-lapse imaging. Post-mitotic cells displayed no fluorescent signal but gradually fluoresced red. When cells entered S phase (square), the cells fluoresced green and underwent mitosis. The time course was measured in 1-h intervals, except for the last two images (15-min intervals). A time-lapse movie can be seen in Supplementary Video 1. Scale bar: 20 μm. (c) Dot plots of the cell-cycle intervals analysed by time-lapse imaging. By monitoring cell colour, the interval of each cell-cycle phase was analysed. Entry into S phase was indicated by a green fluorescent signal, and the timing of mitosis was indicated by the appearance of a new cell. Values indicate the average interval.