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. Fig. 7C . Values plotted are the ratio of mean loaded MCM of first cell cycle with or without nutlin block and release divided by mean loaded MCM of second cell cycle. Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. Samples were compared by unpaired, two tailed t test. p=0.0036**. e. Percentage of underlicensed cells from Fig. 7C . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. Untreated and nutlin treated cells were compared by unpaired, two tailed t test. First cell cycle and nutlin block p=0.0027**, Nutlin block and second cell cycle p=0.2447 (ns). 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 . h. Loaded MCM in early S phase determined by flow cytometric analysis of cells treated as Fig. 7F , measuring DNA Content (DAPI), Loaded MCM (anti-Mcm2), and DNA Synthesis (EdU). Orange line is first cell cycle, green line is second cell cycle with ↑Cyclin E1 starting at 24 hours after G0 release, and grey line is second cell cycle. See Fig. S4G . i. Comparison of early S phase loaded MCM per cell from Fig. 7F . Values plotted are the ratio mean loaded MCM of second cell cycle divided by mean loaded MCM of first cell cycle. Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. Samples were compared by unpaired, two tailed t test. p=0.0036** j. Percentage of underlicensed cells from Fig. 7F . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. Samples were compared by unpaired, two tailed t test. first cell cycle vs second cell cycle with ↑Cyclin E1 p=0.6593 (ns), second cell cycle with ↑Cyclin E1 vs second cell cycle untreated p<0.001****. The copyright holder for this preprint (which wa