Fig. 4
Cell-cycle fluorescence reporter system FUCCI shows failed replication cycles in TMZ-treated cells. (A) FUCCI uses two fluorescent proteins (green and red) to indicate the position in the cell cycle. Two lineage trees from the control experiment show how the fluorescence signal changes along each branch. A plot below shows an example of the red \(\:R\left(t\right)\) and green \(\:G\left(t\right)\) fluorescence for a cell that successfully divided at \(\:t=35\) h. (B) Histograms of the “cell cycle phase” \(\:\varphi\:\) calculated as \(\:\varphi\:=G\left(t\right)/(G\left(t\right)+R\left(t\right))\). Between 0.5k and 4.3k cells have been analysed. A peak close to zero represents cells in the G1 phase, whereas a peak close to 1 represents cells in the G2/M phase. As time progresses, the control population develops a third, smaller peak close to \(\:\varphi\:\approx\:0.7\) (arrow), whereas treated cells do not exhibit this peak. (C-F) Average red and green fluorescence along trajectories that end up with a division (panels C, E) or cell death/detachment (panels D, F), for the control and 500 µM TMZ. The time before division is calculated as \(\:t-{t}_{0}\), where \(\:t\) is the time at which fluorescence was obtained and \(\:{t}_{0}\) is the time of division (\(\:{t}_{0}>t)\), hence the negative values on the horizontal axis. Shaded areas indicate G1 and G2/M phases. The plot for treated cells that went on to divide shows the evidence of failed earlier divisions. In treated cells that did not divide, FUCCI signals vary much more slowly, indicating a possible cell cycle arrest. A single biological replicate has been analysed and only human-verified trajectories have been included (\(\:n=\text{28,35,14,10}\), respectively). Errors = SEM.
