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

s not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/558783 doi: bioRxiv preprint Supplemental Figure 1 . Flow cytometry gating and alternate cell lines a. Flow cytometry gating to isolate cells for analysis. Proliferating RPE1 cells (Fig. 1A) were processed for analytical flow cytometry for chromatin-bound proteins, labeling DNA Content (DAPI), Loaded MCM (anti-Mcm2) and DNA Synthesis (EdU). Cells were labeled with 10 uM EdU for 30 minutes before harvesting. Gating to isolate cells from debris is Forward Scatter Area vs Side Scatter Area, cells gate. Gating to isolate single cells from doublets is DAPI Area vs DAPI Height, singlets gate. Gating to determine cell cycle phase distributions is DNA Content vs DNA Synthesis. Color gating for S-MCM DNA positive (orange), G1-MCM DNA positive (blue) and MCM DNA negative (grey) is on DNA Synthesis vs Loaded MCM using a negative control sample without Mcm2 primary antibody or EdU, but with Donkey anti Mouse-488 secondary antibody and 647-azide as a measure of background staining. b. Cell cycle phase of RPE1 cells G0 synchronized by contact inhibition, Fig. 1C , RPE1 cells G0 synchronized by mitogen starvation, Fig. S1I , Wi38 cells G0 synchronized by contact inhibition, Fig. S1G , and NHF1-htert cells G0 synchronized by contact inhibition, Fig. S1E . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. c. Immunoblot for Cyclin D1 or p27 on total protein lysate from RPE1 cells synchronized in G0 or released into the first cell cycle (24 hours) as in Fig. 1C . d. Percentage of S phase cells defined by analytical flow cytometry. RPE1 cells were synchronized in G0 by contact inhibition were released into the cell cycle with 1 uM EdU at time of release, harvesting cells, 24, 28, and 32 hours after release from G0. S phase was determined by DAPI (DNA Content) and EdU (DNA Synthesis) cells as in Fig. S1A . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. e. Flow cytometry of chromatin-bound protein on Wi38 cells synchronized in G0 by contact inhibition in 0.1% FBS for 72 hours and released from G0 into the cell cycle, harvesting 24 hours after release (first cell cycle) and 48 hours after release (second cell cycle). Fig. 2C . b. Diagram of experiment. For 1xG0: RPE1 cells were synchronized in G0 by contact inhibition for 48 hours, then released into the cell cycle with 5 nM gemcitabine, harvesting cells 24 hours after release. For 3xG0: RPE1 cells were synchronized in G0 by contact inhibition for 48 hours, released into the cell cycle without gemcitabine and grown to confluency to repeat the G0 by contact inhibition a second time, then repeating once again for a 3 rd G0. Then cells were released into the cell cycle with 5 nM gemcitabine, harvesting cells 24 hours after release. c. Flow cytometry of chromatin-bound protein from cells treated as in Fig. S2B , measuring DNA Content (DAPI) and γH2AX (anti H2AX phospho S139). Histograms plot γH2AX of Mid-S phase cells as described in Fig 2C. Upper panel is 1xG0, lower panel is 3xG0. Grey lines are first cell cycle untreated, red lines are first cell cycle treated with 5 nM gemcitabine. d. Percentage of replication stress-induced γH2AX in 1xG0 and 3xG0 release into the first cycle from Fig. S2C . Horizontal bars indicate means, error bars mark standard deviation (SD), n=3 biological replicates. 1xG0 and 3xG0 compared by unpaired, two tailed t