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

Cells stained with EdU were incubated with 10 µM EdU for 30 minutes before harvesting, except in Fig S1D, when cells were incubated with 1 µM EdU from restimulation to harvesting. Cells were harvested with trypsin to measure DNA loaded proteins by flow cytometry. Cells were washed once with PBS, centrifuged at 2,000 x g for 3 minutes, then supernatant was aspirated and pellets lysed on ice in cold CSK with 0.5% triton X-100 and protease and phosphatase inhibitors for 5 minutes. After incubation, 1% Bovine serum albumin (Fisher) in PBS (B-PBS) was added to each sample, mixed, and centrifuged at 2,000 x g for 3 minutes. Supernatant was aspirated, pellets were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 minutes at RT. Then B-PBS was added, mixed, and samples centrifuged (2,000 x g for 7 minutes, all following steps). Supernatant was aspirated, B-PBS was added, and samples stored at 4°C before labeling.