All imaging data was quantified in Volocity (PerkinElmer). To find dynamic changes in GFP intensities, cell nuclei were tracked using the LSS2-mKate-PCNA signal and the Shortest Path algorithm. A maximum distance of 9 μm between nuclei in consecutive timeframes was used to eliminate erroneous tracks. For p27Kip1-GFP, CyclinE1-GFP, and CyclinA2-GFP, nuclear levels of protein were quantified since all three proteins had an exclusively nuclear localization during G1 and S. In each cell, the average value of pixel intensities was calculated, and average background fluorescence was subtracted. All fluorescence intensities were normalized to peak levels. Timing of S-phase entry was determined manually from PCNA fluorescence images. Specifically, S-phase entry is defined by a sharp increase in PCNA intensity, often coinciding with the appearance of nucleoli.
For CDK2L-GFP and DHB-Ven quantification, nuclear intensity was measured by taking a region of interest (ROI) inside the nucleus, and cytoplasmic intensity was measured as the ring region around the nucleus. Cdk2 activity was then measured as the ratio between the ring GFP intensity to the nuclear GFP intensity.
Additional experimental procedures are provided inSupplemental Information .
For CDK2L-GFP and DHB-Ven quantification, nuclear intensity was measured by taking a region of interest (ROI) inside the nucleus, and cytoplasmic intensity was measured as the ring region around the nucleus. Cdk2 activity was then measured as the ratio between the ring GFP intensity to the nuclear GFP intensity.
Additional experimental procedures are provided in
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