CpG-siRNA strategy allows for targeted delivery of Stat3 siRNA into TLR9-positive Cbfb-MYH11/Mpl⁺leukemic cells. (A) Western blot analysis showing constitutive activation of Stat3 in CMM⁺ AML cells compared with untreated or IL-6–treated RAW 264.7 macrophages; β-actin was used as an internal loading control. (B) TLR9 expression in CMM⁺ AML cells. Tlr9 messenger RNA and protein were assessed by using quantitative polymerase chain reaction (qPCR) (left panel) and flow cytometry (right panel). RAW 264.7 macrophages and CD3⁺ T cells were used as positive and negative controls, respectively. (C) Dose- and time-dependent internalization of naked CpG-Stat3 siRNA by CMM⁺ cells. Both molecules were labeled with Cy3 fluorochrome on the 5′ end of the siRNAs to follow their intracellular uptake. AML cells were incubated with various concentrations of fluorescently labeled CpG-Stat3 siRNA^Cy3 conjugate or unconjugated Stat3 siRNA^Cy3 for indicated times without any transfection reagents. Percentages of Cy3⁺ AML cells were assessed by fluorescence-activated cell sorter.