Genetic inhibition of BTK inhibits cell proliferation in AML cells. AML cell lines (TF-1 and U937) were transduced with BTK-targeted miRNA GFP-tagged lentiviral constructs. (A) Transfected cells were measured for GFP expression using flow cytometry for 2 BTK-targeted miRNA (BTK437 and BTK1092) in TF-1 cells. RNA was extracted from TF-1 and U937 cells transduced with BTK-targeted and nonsilencing miRNA control constructs and examined for BTK expression by RT-PCR at the indicated times. mRNA expression was normalized to GAPDH mRNA levels. (B) Protein extracts were also obtained and western blot analysis was conducted for p-BTK and BTK protein levels. (C) TF-1 and U937 were transduced with either BTK-targeted miRNA or nonsilencing control miRNA construct for 72 hours. Cell number was assessed by Cell TitreGlo assay. (D) AML blasts, AML cell lines, and CD34⁺ HSCs were transduced with BTK-targeted miRNA and control miRNA constructs, and colony-forming assays were performed to show the number of colonies detected. In all panels, the values indicate the mean ± SD from 3 independent experiments. *Statistical significance of P < .05 between the different treatment groups.