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

To synchronize RPE1 cells in G0 by contact inhibition, cells were grown to 100% confluency, washed with PBS, and incubated in DMEM with 10% FBS and 2 mM Lglutamine for 48 hours. To release cells into the first or second cell cycles, G0 cells were re-stimulated by passaging 1:10 for the first cycle, harvesting 24 hours later or 1:20 for the second cycle, harvesting 48 hours later, in DMEM with 10% FBS and 2 mM L-glutamine. To synchronize RPE1 cells in G0 by serum starvation, cells were plated sub-confluent, washed 3 times with PBS, and starved in DMEM with 0% FBS and 2 mM L-glutamine for 72 hours. To release cells into the first or second cell cycles, cells were washed with PBS and re-stimulated by washing once with PBS and adding DMEM with 10% FBS and 2mM L-glutamine for 24 hours (first cycle) or 48 hours (second cycle). To synchronize NHF1-hTERT cells in G0, cells were grown to 100% confluency, washed with PBS, and incubated in DMEM with 0.1% FBS and 2 mM L-glutamine for 72 hours. To release cells into the first or second cell cycles, G0 cells were re-stimulated by passaging 1:4 for first cycle, harvesting 24 hours later or 1:8 for second cycle, . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 harvesting 48 hours later, in DMEM with 10% FBS and 2 mM L-glutamine. To synchronize Wi38 cells in G0, cells were grown to 100% confluency, washed with PBS, and incubated in MEM with 0.1% FBS, 2 mM L-glutamine, and NEAA for 72 hours. To release cells into the first or second cell cycles, G0 cells were re-stimulated by passaging 1:4 for first cycle, harvesting 24 hours later or 1:8 for second cycle, harvesting 48 hours later, in in MEM with 10% FBS, 2 mM L-glutamine, and NEAA.