Stat3 phosphorylation is constitutive and inducible in primary pediatric AML samples and correlates with survival. (A) Primary AML samples from the COG AML Reference Laboratory were analyzed by FACS for pStat3. The percentage of pStat3⁺ events in the unstimulated condition is shown. Samples with more than or equal to 20% of pStat3⁺ cells were considered to have constitutive Stat3 activation. BMMCs are from normal donors. (B) The fold change in the percentage of pStat3⁺ cells after stimulation with 100 ng/mL G-CSF, compared with the unstimulated cells, is shown for the 18 primary AML samples from COG and the 5 normal control samples. (C) Total Stat3 protein levels for the NK samples and selected normal BMMC samples were measured by Western blot. The band density for each Stat3 band (including Stat3α/β doublets) was normalized to the corresponding β-actin band density. This value was then divided by the normalized density for a KG-1 sample run on the same gel (not shown) to adjust for differences in exposures between gels. The adjusted densitometry values for each band are shown at the bottom. The figure was compiled from nonadjacent lanes and/or separate gels, with the exception of lanes 1 and 2 (samples NK1 and NK2), which were adjacent on the same gel. (D) The fold change for cells stimulated with G-CSF at the indicated doses, compared with unstimulated cells, is shown for 5 primary AML samples obtained from the Texas Children's Cancer Center.