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

test. p=0.153 (ns). e. Comparison of γH2AX intensity per cell presented as fold-change between gemcitabine-treated and untreated cells from Fig. S2C . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. Samples compared by unpaired, two tailed t test. p=0.1464 (ns). Immunoblot for Cdc6 and Cdt1 on total protein lysate from RPE1 cells constitutively producing either 5myc-Cdc6 WT or 5myc-Cdc6-mut (a mutant of Cdc6 that is not targeted for degradation by APC Cdh1 : R56A, L59A, K81A, E82A, N83A) and a doxycycline inducible Cdt1-HA. RPE1 cells were synchronized in G0 by contact inhibition, treated with 100 ng/mL doxycycline for 4 hours before re-plating cells to release into the first cell cycle, harvesting cells 24 hours after release. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 MCM of second cell cycle. Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. First and second cell cycles compared by unpaired, two tailed t test. p=0.8635 (ns). f. Percentage of underlicensed cells from early S phase cells in Fig. S4D . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. First and second cell cycles compared by unpaired, two tailed t test. Cdc6 WT p=0.0041** Cdc6 mut p=0.0001***. g. Flow cytometry of chromatin-bound protein from cells shown in Fig. 7H Fig. 1G . 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. RPE1-hTert contact p=0.0035** RPE1 starve p<0.001****, Wi38 p<0.001****, NHF1 p=0.006***. 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 . 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 Figure 5 . Cells re-entering the first G1 after G0 lack a licensing checkpoint-induced G1 delay. a. Immunoblot of total protein lysates of cells released from G0 into the first cell cycle with siControl or siCdt1 (24 hours after release) and of proliferating cells treated with siControl or siCdt1 for 72 hours. b. Cell cycle phase distribution of cells treated as Fig. 5A defined by DNA synthesis (EdU) and DNA Content (DAPI) using flow cytometry. Data plotted are mean percentage of G1 (green), S (purple), and G2/M (grey) phases. G1 percentage: 1st cycle siControl = 33%, 1st cycle siCdt1 A = 32.2%, 1st cycle siCdt1 B = 28.7%, proliferating siControl = 56.7%, proliferating siCdt1 A = 67.5%, prolifer