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

Data were acquired primarily on an Attune NxT flow cytometer (Thermo Fisher), with some data acquired on a CyAn ADP flow cytometer (Beckman Coulter). Data was analyzed using FCS Express 6 (De Novo Software). Gates are shown in Fig. S1 . Gate to isolate cells from debris was from Forward Scatter-Area vs Side Scatter Area. Gate to isolate singlets from doublets was from DAPI Area vs DAPI height (Parent gate: cells). Gate to isolate cell cycle phases was from DAPI Area ( Replication stress induced γH2AX was gated as cells equal to or greater than the top 5%-6% of γH2AX signal from untreated cells (Parent gate: Mid S). Each flow cytometry plot typically has 9,000-11,000 total single cells. Histogram counts were normalized to the peak value of the 2 nd cell cycle or siControl. The normalization allows visual comparison of cell distributions between populations with different numbers of cells due to changes in synchrony in the second cell cycle. The quantification of relative loaded MCM or underlicensed cells were not normalized.