Figure 2
Figure 2. Design of the CpG(A)-STAT3 siRNA conjugate for targeting STAT3 in human TLR9+ cells. (A) Structure and sequence of the CpG(A)-STAT3 siRNA: CpG(A) ODN (D19 sequence) was conjugated to the STAT3 siRNA guide (AS) strand through a flexible carbon chain linker; deoxyribonucleotides are underlined; asterisks indicate phosphothioation sites; shown is a position of the fluorochrome (Cy3) at the 5′ end of the STAT3 siRNA passenger strand. (B) Targeted delivery of STAT3 siRNA into various populations of primary human immune cells in vitro. Human PBMCs were incubated in the presence of fluorescently labeled CpG(A)-STAT3 siRNACy3 or unconjugated STAT3 siRNACy3 in various concentrations for the times indicated without any transfection reagents. Percentages of Cy3+CD14+ monocytes, CD303+ (BDCA2+) pDCs, CD1c+ (BDCA1+) mDCs, CD19+ B cells, CD56+ NK cells, and CD3+ T cells were assessed by flow cytometry. Similar results were obtained from 3 independent experiments. (C) CpG(A)-STAT3 siRNA treatment leads to STAT3 gene silencing in various immune cell populations. Monocytes, mDCs, pDCs, and B cells were incubated with 500nM CpG-siRNAs targeting STAT3 or Luc (as negative control) for 18 hours. STAT3 expression was measured using qPCR. Shown are results normalized to TBP gene expression levels from 1 of 3 independent experiments; STAT3 expression level in control CpG-Luc siRNA-treated samples was set as 100%. Data are shown as means ± SEM (n = 3). Statistically significant differences are indicated with asterisks. ***P = .0005; **P = .0034; *P = .03. (D) TLR9 is expressed in target immune cell populations sensitive to CpG(A)-siRNA–mediated gene silencing. TLR9 expression was measured by qPCR in enriched populations of monocytes, T cells, and B cells or in cultured mDCs and pDCs. The results are representative of 2 independent experiments performed in triplicate. Data are shown as means ± SEM. (E) STAT3 silencing by CpG(A)-siRNA conjugates depends on TLR9 targeting and activation. STAT3 expression was assessed by qPCR in cultured pDCs treated for 18 hours using CpG(A) alone, CpG(A) linked to nonsilencing control RNA, nontargeting GpC(A)-STAT3 siRNA, or CpG(A)-STAT3 siRNA. Shown are results from 1 of 2 independent experiments performed in triplicate. Data are shown as means ± SEM.

Design of the CpG(A)-STAT3 siRNA conjugate for targeting STAT3 in human TLR9+ cells. (A) Structure and sequence of the CpG(A)-STAT3 siRNA: CpG(A) ODN (D19 sequence) was conjugated to the STAT3 siRNA guide (AS) strand through a flexible carbon chain linker; deoxyribonucleotides are underlined; asterisks indicate phosphothioation sites; shown is a position of the fluorochrome (Cy3) at the 5′ end of the STAT3 siRNA passenger strand. (B) Targeted delivery of STAT3 siRNA into various populations of primary human immune cells in vitro. Human PBMCs were incubated in the presence of fluorescently labeled CpG(A)-STAT3 siRNACy3 or unconjugated STAT3 siRNACy3 in various concentrations for the times indicated without any transfection reagents. Percentages of Cy3+CD14+ monocytes, CD303+ (BDCA2+) pDCs, CD1c+ (BDCA1+) mDCs, CD19+ B cells, CD56+ NK cells, and CD3+ T cells were assessed by flow cytometry. Similar results were obtained from 3 independent experiments. (C) CpG(A)-STAT3 siRNA treatment leads to STAT3 gene silencing in various immune cell populations. Monocytes, mDCs, pDCs, and B cells were incubated with 500nM CpG-siRNAs targeting STAT3 or Luc (as negative control) for 18 hours. STAT3 expression was measured using qPCR. Shown are results normalized to TBP gene expression levels from 1 of 3 independent experiments; STAT3 expression level in control CpG-Luc siRNA-treated samples was set as 100%. Data are shown as means ± SEM (n = 3). Statistically significant differences are indicated with asterisks. ***P = .0005; **P = .0034; *P = .03. (D) TLR9 is expressed in target immune cell populations sensitive to CpG(A)-siRNA–mediated gene silencing. TLR9 expression was measured by qPCR in enriched populations of monocytes, T cells, and B cells or in cultured mDCs and pDCs. The results are representative of 2 independent experiments performed in triplicate. Data are shown as means ± SEM. (E) STAT3 silencing by CpG(A)-siRNA conjugates depends on TLR9 targeting and activation. STAT3 expression was assessed by qPCR in cultured pDCs treated for 18 hours using CpG(A) alone, CpG(A) linked to nonsilencing control RNA, nontargeting GpC(A)-STAT3 siRNA, or CpG(A)-STAT3 siRNA. Shown are results from 1 of 2 independent experiments performed in triplicate. Data are shown as means ± SEM.

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