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

. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 5'-CCUACGUCAAGCUGGACAATT-3' For siRNA transfections, siRNA was spotted into plates with DharmaFECT 4 transfection reagent (Dharmacon) and Opti-MEM (Gibco), incubating for 20 minutes before adding cells in DMEM with 10% FBS and 2 mM L-glutamine (final concentrations), harvesting cells 72 hours later for proliferating experiments, and 24 hours later for G0 release, as described above. To overproduce Cyclin E1 or Cdt1-HA, 100 ng/mL of doxycycline (CalBiochem) was added at times indicated in the figure legend. To block and release cells with nutlin-3a (Sigma Aldrich), RPE1 were synchronized in G0 by contact inhibition, released into the first cell cycle, adding 10 uM nutlin-3a 10 hours after release, washing off nutlin-3a 18 hours after release by washing 3 times with PBS and adding back DMEM with 10% FBS and 2 mM L-glutamine. To treat cells with gemcitabine (Sigma Aldrich) in Fig. 2 , cells were synchronized in G0 by contact inhibition, released into the first cycle and treated with 50 nM gemcitabine from 22-24 hours or released into the second cycle and treated with 50 nM gemcitabine from 44-46 hours. To treat cells with gemcitabine in Fig. S2 , cells were synchronized in one or three G0 (above), adding 5 nM gemcitabine at time of re-stimulation from 0-24 hours.