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

The supplemental figures all support the main figures. Figure S1 defines flow cytometry gating and raw data for additional cell lines summarized in Figure 1G and H. Figure S2 demonstrates replication stress sensitivity over repeated rounds of G0 and cell cycle reentry. Figure S3 is complete flow cytometry color dot plots and histograms of G1 MCM loading with siCdt1 treatment. Figure S4 is complete flow cytometry color dot plots for the nutlin-3a block and release as well as Cyclin E1 overproduction in the second cell cycle. It also includes the minimal effects of Cdc6 and Cdt1 overproduction in the first cell cycle on underlicensing. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/558783 doi: bioRxiv preprint replicates. First and second cell cycles were compared by unpaired, two tailed t test. RPE1-hTert contact p=0.0035** RPE1 starve p<0.001****, Wi38 p<0.001****, NHF1 p=0.006***. Figure 2 . The first S phase after G0 is hypersensitive to replication stress. a. Diagram of experimental workflow. RPE1-hTert cells were synchronized in G0 by contact inhibition and released into the first cell cycle (24 hours) or second cell cycle (48 hours). Cells were treated with 50 nM gemcitabine or vehicle for the last 2 hours before harvesting for flow cytometry. b. Flow cytometry of chromatin-bound proteins in cells treated as in Fig. 2A and stained for DNA content (DAPI), and γH2AX (anti-H2AX phospho S139). Red cells are replication stress-induced γH2AX-positive (gemcitabine), as indicated in Fig. 2C . See also Fig. S2 for DNA-loaded MCM in these same cells. c. Gating of cells from Fig. 2B to define the threshold of γH2AX signal induced by gemcitabine. The rectangle isolates mid-S phase cells. Inset: Mid-S phase cells in which replication stress-induced γH2AX-positive cells (red) are defined as those equal to or above the top 5% of the corresponding untreated cells; these cells were also marked red in Fig. 2B . d. Percentage of replication stress-induced γH2AX in the first and second cell cycles from Fig. 2B . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. Untreated and gemcitabine-treated cells were compared by unpaired, two tailed t test. First cell cycle p=0.0035**; second cell cycle p=0.1174 (ns). e. Histograms of mid-S phase γH2AX intensity per cell from Fig. 2B ; the upper panel is the first cell cycle and the lower panel is the second cell cycle. Grey lines are untreated cells, red lines are gemcitabine-treated cells. f. Comparison of γH2AX intensity per cell presented as fold-change between gemcitabine-treated and untreated cells from cells in Fig. 2E . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. First and second cell cycles were compared by unpaired, two tailed t test, p=0.0023**. Model. Cdt1 depletion causes slow origin licensing leading to p53-dependent inhibition of CDK2 which delays S phase entry. b. Immunoblot of the indicated endogenous proteins in total protein lysates of RPE1-hTert p53 WT or p53 null, (knockout "KO") cells treated with siControl, siCdt1 A, or siCdt1 B for 72 hours. c. Loaded MCM in early S phase determined by flow cytometric analysis of cells treated as in Fig. 4B Fig. 4C . Values plotted are the ratio of mean loaded MCM in cells treated with siCdt1 and cells treated with siControl as indicated. Horizontal bars indicate means, error bars