Figure 2
Figure 2. CpG-STAT3dODN inhibits STAT3 DNA binding, tyrosine phosphorylation, and transcriptional activity in target immune and leukemic cells in vitro. (A) CpG-STAT3dODN inhibits DNA binding of STAT3 and STAT1 but not other transcription factors (TFs). Mouse splenocytes or human KG1a leukemic cells were incubated with indicated CpG-STAT3dODN, control CpG-scrODN (scrambled decoy), or CpG-mutODN (point-mutated decoy) at 500 nM for 48 hours; CpG(A) = D19, CpG(B) = 1668. Cells were then stimulated as indicated or left untreated before isolation of nuclear extracts. The DNA-binding activity of various transcription factors was assessed in EMSA using radiolabeled probes specific for STAT1/STAT3, STAT5, and NF-κB; band identities were verified using specific antibodies; *, unspecific bands. Results represent at least 2 independent experiments with similar outcome. (B-C) CpG-STAT3dODN dose-dependently reduces transcriptional STAT3 activity. MV4-11 (B) and KG1a (C) AML cells were incubated for 18 hours with indicated doses of CpG-STAT3dODN or control CpG-scrODN and then transfected with STAT3-responsive α2M-promoter-luciferase reporter plasmid or empty vectors, together with Renilla-luciferase plasmid to control transfection efficiency. After 30 hours, the level of STAT3-induced transcriptional activity was assessed in cell lysates by measuring dual-luciferase activity in 2 independent experiments in triplicates; means ± standard error of the mean (SEM) (n = 3). (D-E) CpG-STAT3dODN reduces STAT3 activation but not protein levels in AML cells. (D) Levels of pSTAT3 and total STAT3 protein in human MV4-11 AML cells incubated for 48 hours with 500 nM CpG(D19)-STAT3dODN in comparison with CpG(D19)-scrODN or GpC-STAT3dODN used as negative control. Representative western blotting results showing pSTAT3 and STAT3 protein levels, using β-actin to control loading. (E) Dose-dependent inhibition of STAT3 phosphorylation by CpG-STAT3dODN. Mouse splenocytes were incubated with different concentrations of CpG(1668)-STAT3dODN or CpG(1668)-scrODN control for 48 hours, then stimulated for 20 minutes using IL-6 (20 ng/mL). pSTAT3 levels were assessed using intracellular staining with specific antibodies and flow cytometry. Representative histogram overlay (top) and averaged mean fluorescence intensities (MFIs) from triplicate samples (bottom); shown are means ± SEM (n = 3). GM-CSF, granulocyte macrophage colony-stimulating factor; LPS, lipopolysaccharide.

CpG-STAT3dODN inhibits STAT3 DNA binding, tyrosine phosphorylation, and transcriptional activity in target immune and leukemic cells in vitro. (A) CpG-STAT3dODN inhibits DNA binding of STAT3 and STAT1 but not other transcription factors (TFs). Mouse splenocytes or human KG1a leukemic cells were incubated with indicated CpG-STAT3dODN, control CpG-scrODN (scrambled decoy), or CpG-mutODN (point-mutated decoy) at 500 nM for 48 hours; CpG(A) = D19, CpG(B) = 1668. Cells were then stimulated as indicated or left untreated before isolation of nuclear extracts. The DNA-binding activity of various transcription factors was assessed in EMSA using radiolabeled probes specific for STAT1/STAT3, STAT5, and NF-κB; band identities were verified using specific antibodies; *, unspecific bands. Results represent at least 2 independent experiments with similar outcome. (B-C) CpG-STAT3dODN dose-dependently reduces transcriptional STAT3 activity. MV4-11 (B) and KG1a (C) AML cells were incubated for 18 hours with indicated doses of CpG-STAT3dODN or control CpG-scrODN and then transfected with STAT3-responsive α2M-promoter-luciferase reporter plasmid or empty vectors, together with Renilla-luciferase plasmid to control transfection efficiency. After 30 hours, the level of STAT3-induced transcriptional activity was assessed in cell lysates by measuring dual-luciferase activity in 2 independent experiments in triplicates; means ± standard error of the mean (SEM) (n = 3). (D-E) CpG-STAT3dODN reduces STAT3 activation but not protein levels in AML cells. (D) Levels of pSTAT3 and total STAT3 protein in human MV4-11 AML cells incubated for 48 hours with 500 nM CpG(D19)-STAT3dODN in comparison with CpG(D19)-scrODN or GpC-STAT3dODN used as negative control. Representative western blotting results showing pSTAT3 and STAT3 protein levels, using β-actin to control loading. (E) Dose-dependent inhibition of STAT3 phosphorylation by CpG-STAT3dODN. Mouse splenocytes were incubated with different concentrations of CpG(1668)-STAT3dODN or CpG(1668)-scrODN control for 48 hours, then stimulated for 20 minutes using IL-6 (20 ng/mL). pSTAT3 levels were assessed using intracellular staining with specific antibodies and flow cytometry. Representative histogram overlay (top) and averaged mean fluorescence intensities (MFIs) from triplicate samples (bottom); shown are means ± SEM (n = 3). GM-CSF, granulocyte macrophage colony-stimulating factor; LPS, lipopolysaccharide.

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