CpG-siRNA uptake and gene silencing in TLR9⁺ MM and AML cells. (A) The majority of tested MM (left) and AML (right) cells expressed high levels of TLR9 protein. Representative results from at least 2 Western blotting analyses are shown; positions of TLR9 and β-actin for loading control are indicated. (B) CpG-siRNA is quickly internalized by established MM and AML cells as well as by patient AML blasts in vitro. Cells were incubated with 500nM FITC-labeled CpG-STAT3 siRNA for 1 hour. The percentage of FITC⁺ cells was analyzed by flow cytometry. (C-D) CpG-STAT3 siRNA accumulated in early endosomes shortly after intracellular uptake. KMS-11 myeloma cells (C) and MV4-11 leukemia cells (C-D) were treated with 500nM CpG-STAT3 siRNA^FITC for 1 hour. The intracellular localization of the conjugate was assessed by confocal microscopy. Green indicates CpG-STAT3 siRNA^FITC; red, EEA1 (early endosome marker); and blue, nuclear staining with DAPI. (E) Target gene silencing in cultured KMS-11 cells incubated with 500nM CpG-STAT3 siRNA for 18 hours. Shown are results averaged from 4 independent experiments analyzed by qPCR and normalized to GAPDH.