Figure 1. Characterization of cell cycle in HT1080 FUCCI cells.

The FUCCI system (a) consists of two fluorescent polypeptides that are ubiquitylated and degraded by proteasomes in a cell cycle regulated manner. The red probe (mKO-hCdt1) increases in G1-phase and is degraded at the onset of S-phase. The green probe (mAG-hGeminin) increases at the onset of S-phase and is degraded upon anaphase of mitosis. (b,c) Untreated cells tracked from birth. As cells progress into S-phase, green signal accumulates and red signal continues to degrade while the green signal continues to increase. Absence of red signal corresponds to the transition to late S-phase. Cells remain green through late S- and G2-phase and enter mitosis after 14–18 h as indicated by observable breakdown of the nuclear envelope, and the green signal is rapidly degraded at anaphase onset (~15 h in this example). (c) FUCCI trace representing control cells measured from birth to mitosis. The average cell cycle time is ~16 h. Cells spend ~4–6 h in G1-phase, ~8–10 h in S/G2-phase, and ~1.0 h in mitosis. Bar is 10 μm. (d–f) Cell cycle arrest standards. (d) A representative FUCCI trace of cell born into the G1-phase arrestor, Cdk4/6 inhibitor, PD-0332991. Cells progress into G1-phase (red) and remain until the end of acquisition (60 h). (e) A representative FUCCI trace of cell treated with S-phase arrestor, DNA polymerase-α inhibitor, aphidicolin. Cells tracked from G1-phase (red), progress into S-phase with normal kinetics and become green. Cells remain green until the end of acquisition (45 h). (f) A representative FUCCI trace of cell treated with the G2-arrestor, Cdk1 inhibitor, 20 μM RO-3306. Cells tracked from G1-phase (red), progress normally into S-phase with normal kinetics and become green. Cells remain green until the end of acquisition (70 h). See Supplementary Fig. 1a–d online for FUCCI distribution over time. Supplementary videos S1, S2, S4, S5 online. Cell number tracked: untreated, 22, PD0332991, 19, aphidicolin, 24, RO-3306, 20.