FIG. 6.

cSCK-pa₁₀₀ mediated silencing of iNOS expression by siRNA. Anti-iNOS siRNA481 (100 nM) was transfected into mouse monocyte-macrophage RAW264.7 cells using PAEA₁₂₈-b-PS₄₀ cSCK-pa₁₀₀ at different N/P ratios (for N/P=1, cSCK 0.62 μg/mL), Lipofectamine 2000 (1.7 μg/mL), ExGen 500 (18.2 μM), or Polyfect (6.8 μg/mL). After 6 hours iNOS expression was induced by treatment with LPS (lipopolysaccharides from Escherichia coli 055:B5) and γ-IFN (gamma interferon). After 24 hours the supernatant was analyzed for NO production by the Griess assay, and the cells were analyzed for iNOS production by western blotting. (A) Griess assay; (B) western blotting, lane 1, cSCK (6.2 μg/mL), no siRNA; lane 2, 100 nM AllStars Negative Control siRNA with cSCK (6.2 μg/mL); lane 3, 100 nM iNOS siRNA481 with cSCK (6.2 μg/mL); lane 4, 100 nM iNOS siRNA481 with Lipofectamine 2000 (1.7 μg/mL); lane 5, 100 nM iNOS siRNA481 with Polyfect (6.8 μg/mL); lane 6, 100 nM iNOS siRNA481 with ExGen 500 (18.2 μM); lane 7, 100 nM iNOS siRNA481 alone, without any transfection agent.