Increased STAT3 and STAT5 activity in human AML stem/progenitor cells and inhibition by AZD1480 treatment. (A) AML, PBSCs, and CB CD34⁺ cells were evaluated for phosphorylated and total STAT3/5 and actin by western blotting. (B) AML CD34⁺ cells treated with 0.5 µM AZD1480 or DMSO for 30 minutes were evaluated for pSTAT3/5 by flow cytometry. Representative plots are shown. (C-D) Expression of pSTAT3 and pSTAT5 measured by flow cytometry in 4 cord blood and 48 primary AML CD34⁺ samples organized by cytogenetic risk category, represented as log₂(MCF pSTAT3/5/MCF isotype control). (E) Expression of pSTAT3/5 in AML CD34⁺ cells by FLT3-internal tandem duplication (ITD) mutation status. (F-G) Fractional inhibition of pSTAT3/5 in 46 AML CD34⁺ cells organized by cytogenetic risk category exposed to 0.1 µM AZD1480 for 30 minutes. Inhibition was calculated as 1 − (MCF pSTAT3/5-treated cells/MCF pSTAT3/5-untreated cells). (H) Fractional inhibition of pSTAT3/5 inhibition in AML CD34⁺ cells by FLT3-ITD mutation status. (I-J) Correlation of pSTAT3/5 expression (log₂) in AML CD34⁺ cells with pSTAT3/5 inhibition by AZD1480. Results represent mean ± SEM. Significance values: *P < .05, **P < .01, ***P < .001.