Figure 5.

BTG3 is required for the maintenance of G2 arrest after IR. (A) Immunoblots demonstrating BTG3 knockdown after siRNA transfection in HCT116 cells. (B) Flow cytometry analysis of control and BTG3-ablated HCT116 cells before and after IR. Early exit from the G2 block was observed in the BTG3 knockdown cells. (C) Quantification of results from three independent experiments shown in panel B. The G2 to G1 ratio was used to represent the degree of G2 arrest. ^*Indicates statistical significance (P<0.05) as determined by Student's t-test. (D, E) Disregulation of Cyclin B-associated H1 kinase activity in BTG3 knockdown cells after IR. HCT116 (D) or LNCaP (E) cells were first transfected with siRNA then irradiated with IR as in panel B. Cyclin B–Cdk1 kinase activity was determined by immunoprecipitation with anti-Cyclin B antibody, followed by an in vitro kinase assay using histone H1 as substrate. The H1 kinase activity was quantified and presented as bar graphs. (F) The G2/M checkpoint response is enhanced and extended in BTG3-overexpressing HCT116 cells. Cells were transfected with HA-BTG3 and irradiated with IR 24 h after the transfection. The G2/G1 ratios are shown in parentheses. Similar results were observed in two other independent experiments.