Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Jacob Peter Matson; Amy M. House; Gavin D. Grant; Huaitong Wu; Joanna Perez; Jeanette Gowen Cook

b. An individual cell trace of mean nuclear Cdc6 intensity imaged from Fig 6A. The trace is orange for the first cell cycle and grey for one daughter in the cell second cell cycle. Hours indicate hours from G0 release beginning at 6.5 hours after release. Circles indicate relevant features: rise is first appearance of nuclear Cdc6; peak is maximum nuclear Cdc6 before S phase and cytoplasmic translocation. The "licensing window" is the difference between peak and rise. Division is the last image before cytokinesis. c. Quantification of licensing window time (Cdc6 peak time minus Cdc6 rise time) for the 50 cells imaged in Fig. 6A , two complete cell cycles for each cell were analyzed. d. Timing of Cdc6 peak relative to G0 release (first cell cycle) or to division (second cell cycle) for the 50 cells imaged in Fig. 6A , two cell cycles for each cell. e. Ratio of licensing window time divided by Cdc6 peak time for each cell imaged as in Fig. 6A , 50 cells total with both first and second cell cycle f. Ratio of mean cytoplasmic DHB-mCherry divided by mean nuclear DHB-mCherry at the time of Cdc6 peak for the 50 cells imaged in Fig. 6A , two cell cycles for each cell. f. Diagram of experiment. RPE1 cells containing integrated doxycycline inducible exogenous Cyclin E1 were synchronized in G0 by contact inhibition, then released into the cell cycle, adding 100 ng/mL of doxycycline at 24 hours after release to overproduce Cyclin E1 and shorten G1 of the second cell cycle, harvesting cells at 40 hours after release from G0. Untreated cells were harvested at 24 hours (first cell cycle) and 40 hours (second cell cycle) after release from G0. g. Immunoblot for Cyclin E1 on total protein lysate from cells treated as Fig. 7F .