Fig 2. p27^Kip1 is required to prevent S-phase entry after DNA damage.

(A) Depletion of p27^Kip1 leads to prolonged CDK2 activity after IR treatment. MCF7 cells were transfected with non-targeting control or p27 siRNAs for 72h before treatment with 6 Gy of IR. Cells were harvested at the indicated time points post IR and divided in 2. The first half was used to measure CDK2 kinase activity in vitro after immunoprecipitation using histone H1 as a substrate. The kinase assay reactions for the Control and the p27 siRNA samples were ran on 2 different gels but exposed on the same film for the same time duration. The second half of the RNAi transfected cells was used for Western blotting analysis with the indicated antibodies (left). (B) Cell depleted of p27^Kip1 do not arrest in G1 and progress into S-phase after IR treatment. MCF7 cells were transfected with non-targeting control or p27 siRNAs for 72h before treatment with 0 or 6 Gy of IR. Cell were labeled with BrdU at the indicated time points and then fixed and stained with propidium iodide (PI). The percentage of cells in G1 at the indicated time points was measured by flow cytometry analysis of PI incorporation and (C). The percentage of cells in S-phase at the indicated time points was measured by flow cytometry analysis of BrdU incorporation. The data is presented as mean of 3 independent experiments ± SEM.