Fig. 1

Deletion analysis of the upstream 5′-flanking region (−1034 to +88 bp) of the human iNOS gene in PVEC. Designation of constructs is depicted on the left. A complete NF-κB element resides at position −115 to −106 bp. Open bars represent basal expression, and closed bars represent the effect of IL-1β, 10 ng/mL, added to culture medium for 24 hr. Luciferase activity is expressed relative to that achieved with the phiNOS-1034Luc plasmid under basal conditions. Luciferase activity was normalized to a cotransfected β-galactosidase internal standard. Values shown are the means ± SEM of five independent experiments. Fold induction, a measure of IL-1β inducibility of the human iNOS promoter construct, is expressed as the ratio of IL-1β-stimulated activity to basal activity. In a representative experiment, absolute values of luciferase activity in cells transfected with an empty pGL2 vector were 2.5 × 10⁴ ± 0.06 × 10⁴ RLU/OD₄₂₀ × 1hr, and 2.6 × 10⁴ ± 0.03 × 10⁴ RLU/OD₄₂₀ × 1hr in untreated cells and cells treated by IL-1β, respectively. In cells transfected with phiNOS-1034LUC plasmid, corresponding values were 136 × 10⁴ ± 7.3 × 10⁴ RLU/OD₄₂₀ × 1hr, and 419 × 10⁴ ± 23.6 × 10⁴ RLU/OD₄₂₀ × 1hr, respectively. Luciferase activity (expressed in relative luciferace units) was normalized by β-galactosidase activity (expressed as OD₄₂₀ reached after incubation at 37°C for 1hr).