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

ating siCdt1 B = 77.5%. Error bars are SD, n= minimum 3 biological replicates. Cell cycle phase percentages from DNA Content vs DNA Synthesis. c. Immunoblot of total protein lysate of RPE1-hTert WT and p53 KO cells synchronized in G0 or released into the first cell cycle with siControl or siCdt1 (24 hours after release) as indicated. d. Cell cycle phase distribution of cells treated as Fig. 5C 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. Error bars are SD, n= minimum 3 biological replicates. G1 percentage 16.6% (siControl) 17.0% (siCdt1 A) 16.4% (siCdt1B). Cell cycle phase percentages from DNA Content vs DNA Synthesis. Figure 6 . Cdc6 dynamics in the first and second cycles after G0. a. RPE1-hTert cells constitutively expressing Cdc6-mVenus, PCNA mTurq2 and a CDK activity sensor (DHB-mCherry), and synchronized in G0 by contact inhibition and release were imaged every 10 minutes for 72 hours. Representative micrographs are shown marking the time of Cdc6 accumulation, CDK activity increase and S phase entry. Images were brightness/contrast adjusted. Representative cell from n=50 analyzed cells. The scale bar is 10 µm and applies to all images.