Inhibition of Axl activation relieves a block in myeloid differentiation. (A) MV4;11 human FLT3-ITD⁺ AML cell line was treated with Ctrl-Fc or Axl-Fc for 2 days and stained with anti-CD11b antibody conjugated with phycoerythrin (left). Separately, the cells treated with media alone were also stained with the antibody (day 0). The histogram lines for day 0 and Ctrl-Fc (day 2) overlapped indistinguishably. Primary FLT3-ITD⁺ AML patient blasts were treated with Ctrl-Fc or Axl-Fc for 2 days and stained with anti-CD11b antibody conjugated with phycoerythrin (right). Each histogram is the representative of 2 separate experiments. (B) MV4;11 human FLT3-ITD⁺ AML cell line (left) or primary FLT3-ITD⁺ AML patient blasts (right) were treated with Ctrl-Fc or Axl-Fc for 4 days and then NBT reduction was assessed by measuring the absorbance at 570 nm. The graph shows mean + SEM for 3 separate experiments. **P < .01. (C) Primary FLT3-ITD⁺ AML blasts from 3 patients (P1 to P3) were treated with Ctrl-Fc (empty bar) or Axl-Fc (filled bar) for 2 days and the mRNA of the indicated gene was quantified by real-time RT-PCR. Data shown is the fold induction of each gene expression by Axl-Fc when compared with that by Ctrl-Fc (arbitrarily set at 1.0). (D) MV4;11 human FLT3-ITD⁺ AML cell line (top) and primary FLT3-ITD⁺ AML patient blasts (bottom) were treated with Ctrl-Fc or Axl-Fc for the indicated times and phospho-C/EBPα (Ser21) and C/EBPα were detected by immunoblot. Actin was used as a loading control. This is the representative of 2 separate experiments.