Figure 5.
PIP-FUCCI dynamics after DNA damage. a) PIP-mVenus dynamics in control U2OS cells and cells treated with neocarzinostatin (NCS) (200 ng/ml). Upper graphs show PIP-mVenus traces from cells treated with NCS in G1 (left, n = 19) or G2 (right, n = 18). Lower graphs are traces from control cells in G1 (left, n = 18) or G2 (right, n = 22) phases. The traces end when cells enter S phase or mitosis respectively; x axis is experiment time in minutes, time of NCS addition is 0 (arbitrary time point was selected for control cells). b) Quantification of PIP-mVenus decline 120 minutes after NCS treatment in U2OS cells in G1 and G2 phases. (Left) Bars are the ratios of detected versus expected PIP-mVenus levels assuming steady signal in G1 phase and linear increase in G2 phase. (Right) Cartoon showing the principle of this analysis. As a control traces from untreated U2OS cells were analyzed with a randomly selected point of mock treatment (for further details see Materials and Methods). Bar graphs show mean ± standard deviation, Wilcoxon rank sum test, p < 0.0001 (G1 control n = 122, G1 NCS n = 19, G2 control n = 121, G2 NCS n = 14). c) Example micrographs and single cell quantification of PIP-FUCCI reporters in U2OS (i, n = 48) and RPE-hTert (ii, n = 59) cells 48 hrs after treatment with NCS. Dashed line indicates time of NCS addition x axis is time in hours. (iii) histogram from NCS-treated RPE-hTert cells as in ii plotting hours between the end of S phase defined by PIP accumulation and the start of degradation of mCherry-Gem1-110 in the absence of cell division (i.e. “mitosis skipping”) (iv) U2OS cells induced to re-replicate by overproducing full-length CDK-resistant Cdt1 (n = 32).