Priority Research Paper Volume 12, Issue 7 pp 5612—5624

Supraphysiological protection from replication stress does not extend mammalian lifespan

Figure 1. Chk1Tg, Rrm2Tg and Chk1Tg;Rrm2Tg MEFs are protected against replication stress compared to WT MEFs. (A) DNA genotyping results for Chk1 and Rrm2 alleles in MEFs; (B) Western blot showing CHK1 and RRM2 protein levels in MEFs; (C) Proliferation curves for Chk1 and Rrm2 transgenic MEFs. Cells were counted and replated every 3-4 days, in three technical replicates per genotype; (D) Cell cycle distribution of MEFs determined by EdU incorporation and DAPI profiles. At least 7000 cells were quantified per condition using high-content microscopy; (E, F) Quantification of γH2AX intensity in MEFs treated with UCN-01 (E) or HU (F) at indicated concentrations for four hours. At least 7000 cells obtained from two technical replicates were quantified per condition using high-content microscopy. Percentages indicate cells with γH2AX intensity above a threshold of 400 AU, and means are indicated by horizontal black lines for each condition. The control cells are the same for (E) and (F), as the results were obtained from the same experiment. **** = P ≤0.0001; *** = P ≤0.001; ns = P > 0.05. Statistical significance was calculated using the unpaired t-test.