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. Author manuscript; available in PMC: 2019 Mar 27.
Published in final edited form as: Cell Syst. 2018 Jun 13;7(1):17–27.e3. doi: 10.1016/j.cels.2018.05.011

Figure 3. Stochastic Endogenous Replication Stress Events Generate Fluctuating CDK2 Activity.

Figure 3.

(A)Thirty random single-cell traces (green lines) and median trace (black line) of cells aligned to time of APC/CCdh1 inactivation following release from quiescence. The indicated inhibitors or DMSO were added 8 hr following mitogen stimulation, prior to APC/CCdh1 inactivation. Median traces were calculated from the entire cell population (n = 583, DMSO; n = 757, ATMi; n = 673, DNA-PKi; n = 611, ATRi; n = 606, Chk1i; n = 448, Wee1i). Dashed line indicates approximate CDK2 activity plateau determined in the DMSO condition. Cells were computationally gated for cells that increase CDK2 activity out of quiescence.

(B and C) Median traces from (A) are shown on the same graph. One of n = 2 biological replicates.

(D) Example of single-cell analysis of CDK2 S-phase dynamics by drawing a line of best fit (blue line) from the time of S-phase entry to the time point when CDK2 activity (green line) crosses a threshold value of 1.5 and calculating the slope of the best-fit line.

(E) Boxplots of single-cell analysis of S-phase CDK2 activity slope in cells exiting quiescence from (A). Significance values are reported for each condition compared with DMSO control. One of n = 2 biological replicates.

(F) Fluctuation analysis of CDK2 activity in cells treated with DMSO or an ATR inhibitor prior to S phase and computationally aligned to the time of CDK2 activity rise. Lines indicate mean and shaded regions indicate SEM (n = 220 for both conditions). One of n = 2 biological replicates.

(G and H) Fluctuation analysis of CDK2 activity in cells treated with indicated doses of hydroxyurea (HU) or aphidicolin (aphi) and computationally aligned to the time of CDK2 activity rise. Gating was performed to only include cells in late G1/early S phase (−1 to 4 hr relative to APC/CCdh1 inactivation) at the time of drug addition. Lines indicate mean and shaded regions indicate SEM (n = 350 for all conditions). One of n = 2 biological replicates.

See also Figure S4.

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