The effects of constitutively activated FLT3 mutants on end-joining DNA repair are associated with inhibited DNA-PKCs activity.

<p>(A) Immunoblot assay. Whole cell extracts from TF1 cells with stable expression of different forms of FLT3 were analyzed by immunoblot assays with anti-DNA LigIII, DNA-PKCs, Ku70, Ku86 and RAD51 antibodies. β-actin was included to confirm equivalent protein loading. (B) Representative images of the changes for nuclear phosphor-DNA-PKCs /γ-H2A.X co-foci in response to IR treatment. Engineered TF1 cells were irradiated with 1.2 Gy. Cells were collected one hour later for immunofluorescence staining with anti-γ-H2A.X (green) and anti-phosphor-DNA-pK (T2609, red). Nuclei were stained with DAPI (blue). Images were acquired with LSM 510 confocal microscope (Zeiss) with 80X objective and processed by Photoshop (Adobe). (C) Diagram shows changes in the fraction of cells with phosphor-DNA-PKCs/γ-H2A.X co-foci in engineered TF1 cells. (D) Expressing FLT3 mutants does not change the inhibitory effects of SAHA on the formation of RAD51/γ-H2A.X nuclear co-foci in irradiated TF1 cells. Engineered TF1 cells were collected 7 hours later after 1.2 Gy of irradiation, and immunofluorescence stained with anti-γ-H2A.X (green) and anti-RAD51 (red). Nuclei were stained with DAPI (blue). Error bars indicate standard deviation. * indicates significance (P<0.05).