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. Author manuscript; available in PMC: 2010 Apr 1.

Published in final edited form as: Nat Biotechnol. 2009 Sep 13;27(10):925–932. doi: 10.1038/nbt.1564

Uptake and gene silencing by CpG-Stat3 siRNA in vitro. (a) Targeted delivery: splenocytes were incubated for 3 h with CpG-Stat3 siRNA or for 24 h with unconjugated Stat3 siRNA labeled with fluorescein (bottom left panel), without any transfection reagents. Percentage of fluorescein-positive CD11c⁺B220⁻ non-plasmacytoid (mDCs) and CD11c⁺B220⁺ plasmacytoid (pDCs) DCs, F4/80⁺Gr1⁻ macrophages (MACs), B220⁺CD11c⁻ B cells, Gr1⁺F4/80⁻ granulocytes and CD3⁺ T cells was assessed by flow cytometric analysis (see also Supplementary Table 1). Splenic CD11c⁺ DCs express high levels of TLR9. Intracellular staining of TLR9 as shown in fixed splenic DCs by flow cytometry (bottom right panel). Similar results were obtained in two independent experiments. (b) Kinetics of CpG-siRNA internalization: CpG-Stat3 siRNA-FITC is quickly internalized by DCs without transfection reagents. The uptake by DC2.4 cells is analyzed by flow cytometry (top row) and confocal microscopy (two lower rows) after incubation with CpG-Stat3 siRNA-FITC (500 nM) for indicated times (two upper rows) or after 1 h incubation at indicated concentrations (bottom row); results are representative of 3 independent experiments; scale bar = 10 μm. (c) Internalized CpG-Stat3 siRNA colocalizes with TLR9 (two upper rows) and transiently interacts with Dicer (two lower rows). DC2.4 cells were incubated with 500 nM of CpG-Stat3 siRNA for indicated times. Shown are confocal microscopy images; green – CpG-Stat3 siRNA-FITC, red – immunofluorescent detection of endogenous TLR9 or Dicer, blue – nuclear staining with DAPI; scale bar = 10 μm. All confocal imaging studies were performed at least 3 times with similar results and the images acquired were characteristic for the majority of analyzed cells (Supplementary Fig. 4). (d) Dose-dependent gene silencing effects of CpG-Stat3 siRNA, comparing to GpC-Stat3 siRNA at the highest dose, as determined by quantitative real-time PCR in DC2.4 cells. Shown are the results of real-time PCR for Stat3, normalized to GAPDH expression levels. The level of Stat3 expression in CpG-scrambled RNA sample is set as 100%. Shown are means ± SEM from 3 independent experiments analyzed in duplicates. (e) Stat3 silencing is impaired in TLR9-deficient primary myeloid cells (top panel) and DCs (bottom panel). Shown are results of two independent experiments, analyzed in triplicates by real-time PCR; means ± SEM. (f) Stat3 DNA-binding is reduced following 48 h incubation of CpG-Stat3 siRNA, relative to CpG-scrambled RNA, in DC2.4 cells. Shown are results of electrophoretic mobility gel-shift assay using radiolabeled probe specifically bound by Stat3 and Stat1, data representing three independent experiments. Positions of Stat dimers are indicated. Full-length gel is presented in Supplementary Figure 13c.

Uptake and gene silencing by CpG-Stat3 siRNA in vitro. (a) Targeted delivery: splenocytes were incubated for 3 h with CpG-Stat3 siRNA or for 24 h with unconjugated Stat3 siRNA labeled with fluorescein (bottom left panel), without any transfection reagents. Percentage of fluorescein-positive CD11c⁺B220⁻ non-plasmacytoid (mDCs) and CD11c⁺B220⁺ plasmacytoid (pDCs) DCs, F4/80⁺Gr1⁻ macrophages (MACs), B220⁺CD11c⁻ B cells, Gr1⁺F4/80⁻ granulocytes and CD3⁺ T cells was assessed by flow cytometric analysis (see also Supplementary Table 1). Splenic CD11c⁺ DCs express high levels of TLR9. Intracellular staining of TLR9 as shown in fixed splenic DCs by flow cytometry (bottom right panel). Similar results were obtained in two independent experiments. (b) Kinetics of CpG-siRNA internalization: CpG-Stat3 siRNA-FITC is quickly internalized by DCs without transfection reagents. The uptake by DC2.4 cells is analyzed by flow cytometry (top row) and confocal microscopy (two lower rows) after incubation with CpG-Stat3 siRNA-FITC (500 nM) for indicated times (two upper rows) or after 1 h incubation at indicated concentrations (bottom row); results are representative of 3 independent experiments; scale bar = 10 μm. (c) Internalized CpG-Stat3 siRNA colocalizes with TLR9 (two upper rows) and transiently interacts with Dicer (two lower rows). DC2.4 cells were incubated with 500 nM of CpG-Stat3 siRNA for indicated times. Shown are confocal microscopy images; green – CpG-Stat3 siRNA-FITC, red – immunofluorescent detection of endogenous TLR9 or Dicer, blue – nuclear staining with DAPI; scale bar = 10 μm. All confocal imaging studies were performed at least 3 times with similar results and the images acquired were characteristic for the majority of analyzed cells (Supplementary Fig. 4). (d) Dose-dependent gene silencing effects of CpG-Stat3 siRNA, comparing to GpC-Stat3 siRNA at the highest dose, as determined by quantitative real-time PCR in DC2.4 cells. Shown are the results of real-time PCR for Stat3, normalized to GAPDH expression levels. The level of Stat3 expression in CpG-scrambled RNA sample is set as 100%. Shown are means ± SEM from 3 independent experiments analyzed in duplicates. (e) Stat3 silencing is impaired in TLR9-deficient primary myeloid cells (top panel) and DCs (bottom panel). Shown are results of two independent experiments, analyzed in triplicates by real-time PCR; means ± SEM. (f) Stat3 DNA-binding is reduced following 48 h incubation of CpG-Stat3 siRNA, relative to CpG-scrambled RNA, in DC2.4 cells. Shown are results of electrophoretic mobility gel-shift assay using radiolabeled probe specifically bound by Stat3 and Stat1, data representing three independent experiments. Positions of Stat dimers are indicated. Full-length gel is presented in Supplementary Figure 13c.