Figure 2. CDK2 Activity Is Transiently Suppressed by Exogenous Replication Stress.

(A) Median CDK2 traces of cycling MCF10A cells computationally gated for cells in S phase during 1-hr neocarzinostatin (NCS) pulse (n = 250, 200 ng/mL; n = 246, 100 ng/mL; n = 235, DMSO). Gray box indicates the time of NCS pulse. One of n = 2 biological replicates.

(B) Median CDK2 traces of cycling MCF10A cells computationally gated for cells in S phase at the time of aphidicolin 1-hr pulse at the indicated concentrations (n = 300, 1000 nM; n = 336, 500 nM; n = 283, 250 nM; n = 221, 125 nM; n = 260, DMSO). Gray box indicates the time of aphidicolin pulse. One of n = 2 biological replicates.

(C) The percentage change in CDK2 activity measured from the time frame when aphidicolin was added to the time of aphidicolin washout. CDK2 recovery time was calculated as the time after aphidicolin washout for CDK2 activity to recover to half of the level of CDK2 activity in the time point immediately preceding aphidicolin addition. Measurements are single-cell analysis of cells in (B). Mean values ± SEM are shown. One of n = 2 biological replicates. ns, not significant.

(D and E) Median traces of cycling cells computationally gated for cells in S phase during treatment with aphidicolin and the indicated DDR inhibitor (n = 231, ATMi; n = 149, ATRi; n = 254, DNA-PKi; n = 186, Chk1i; n = 158, Wee1i; n = 146, DMSO). Gray box indicates time of drug addition. One of n = 2 biological replicates..

(F) Percentage change from (D) and (E) in CDK2 activity of individual cells treated with aphidicolin and the indicated DDR inhibitor. Percentage change was calculated as the change in CDK2 activity from the frame before drugs were added to the frame at 1 hr following drug addition. Mean values ± SEM are shown. Significance values are reported for each condition compared with DMSO control. One of n = 2 biological replicates.

See also Figure S3.