Fig. 6. RB hypophosphorylation is independent of CDK4 and CDK2 activity.

A HCT116 wild type and p53^−/− cells expressing both CDK2 and CDK4 biosensors were treated as indicated for two days then biosensor localisation was assessed by high content imaging and the activity calculated by determining the ratio of cytoplasmic to nuclear biosensor fluorescence. B HCT116 p21^−/− cells expressing both biosensors were treated and analysed as in (A). For each experiment >2000 cells were analysed. The data are the average of triplicate experiments. C HCT116 cells, either untreated or treated with AZD2811 or 10 μM CDK2 or CDK4 selective inhibitors for two days were harvested and immunoblotted with indicated antibodies. D The indicated HCT116 genotypes were treated for two days with either 0.2 μM AZD2811 or 10 μM CDK2 or CDK4 inhibitors and cell lysates immunoblotted for the indicated markers.