Stat3 phosphorylation is constitutive and inducible in AML cell lines. (A) Representative dot plot illustrating the gating scheme used for analysis of pStat3 by FACS. The first gate is set to include cells and exclude debris based on forward and side scatter properties (not shown). The pStat3⁻ and pStat3⁺ gates are based on the isotype control-stained sample (left). The percentages of constitutive and inducible pStat3⁺ cells are determined for unstimulated cells (middle) and cells treated with 100 ng/mL G-CSF for 15 minutes (right), respectively. (B) The percentage of pStat3⁺ cells in the unstimulated condition is shown for all 7 AML cell lines evaluated. Values are mean ± SEM for 5 or more replicates. (C) The fold change in the MFI of cells after stimulation with the indicated dose, compared with the unstimulated cells, is shown for the AML cell lines. A response to G-CSF was defined as an increase of at least 2-fold. Values are mean ± SEM for 2 to 5 replicates.