Figure 4. EVI1 overexpression leads to accumulation of cells in G0/1 phase. (A) Parental U2OS cells, uninduced (-TET) and induced (+TET) U2OS cells conditionally overexpressing EVI1-HA were cultured for 72 h. After 7-AAD staining, the DNA content was measured by flow cytometry. (B) Lysates of parental U2OS cells, uninduced (-TET) and induced (+TET) U2OS cells conditionally overexpressing EVI1 were prepared at the indicated time points after transgene induction, followed by immunoblotting for the indicated proteins. (C) U2OS cells conditionally overexpressing EVI1-HA were induced to express the transgene (+TET) or left uninduced (-TET) for 72 h, followed by immunoprecipitation of Cdk2 from 50 µg protein per lane and an in vitro kinase assay using histone H1 as a substrate to measure Cdk2 activity (upper right panel). Negative controls included induced cells where the Cdk2 antibody or lysate was omitted during immunoprecipitation, or the immunoprecipitate was heated to 95°C for 5 min (upper left panel, from left to right). Immunoblots for HA and actin were prepared in parallel, showing induction of EVI1-HA after 72 h (lower panel). (D) The in vitro kinase assay of Cdk2 activity as described in (C) was reproduced using U2OS cells retrovirally transduced to conditionally overexpress EVI1, with the minor modification that 100 µg protein per lane was used for immunoprecipitation, and that the immunoblot used to demonstrate transgene overexpression was performed with an antibody to EVI1.