FLT3i AC220 downregulated HR and D-NHEJ proteins and inhibited HR and D-NHEJ activity. (A) DSB repair pathways and some key proteins. (B) Western blot analysis of the indicated proteins in parental BaF3 cells transfected with empty plasmid (Empty) and in those expressing FLT3(ITD) after a 24-hour incubation with 10 nM AC220 or vehicle in the presence of IL-3. Cells were nucleofected with linearized plasmids containing HR (C) or D-NHEJ (D) reporter cassette and pDsRed plasmid (transfection efficiency control). HR or D-NHEJ event restores functional GFP expression. After 72 hours, GFP⁺/DsRed⁺ cells in DsRed⁺ cells were analyzed by flow cytometry to assess HR and D-NHEJ activity in untreated (Control) and AC220-treated (10 nM) REH cells and MV-4-11 cells. Results represent mean (percentage) ± SD of GFP⁺/DsRed⁺ cells in DsRed⁺ cells from triplicates per sample. *P = .02, **P < .001, 2-tailed Student t test.