Effect of L-NMMA on the induction of the wild-type and mutated GATA motifs of the Epo promoter/enhancer with Lucreporter constructs in Hep3B cells
| . | % O2 . | Experiment 1 . | Experiment 2 . | Experiment 3 . | Fold induction (mean ± 1 SD) . | |||
|---|---|---|---|---|---|---|---|---|
| RLU/CAT . | Fold induction . | RLU/CAT . | Fold induction . | RLU/CAT . | Fold induction . | |||
| Pwt | 21 | 56.6 | ×50.6 | 75.8 | ×63.4 | 54.3 | ×53.5 | ×55.8 ± 5.5 |
| Pwt | 1 | 2866.4 | 4807.9 | 2902.8 | ||||
| Pwt + L-NMMA | 21 | 42.6 | ×38.0 | 72.9 | ×41.0 | 46.1 | ×20.1 | ×33.0 ± 9.2 |
| Pwt + L-NMMA | 1 | 1619.8 | 2985.7 | 927.0 | ||||
| Pwt + hGATA-2 | 21 | 50.7 | ×24.8 | 80.4 | ×23.4 | 49.4 | ×16.2 | ×21.5 ± 3.8 |
| Pwt + hGATA-2 | 1 | 1257.1 | 1877.8 | 799.8 | ||||
| Pwt + hGATA-2 + L-NMMA | 21 | 57.7 | ×19.1 | 96.3 | ×11.7 | 45.6 | ×11.9 | ×14.2 ± 3.4 |
| Pwt + hGATA-2 + L-NMMA | 1 | 1101.6 | 1130.2 | 544.2 | ||||
| Pm7 | 21 | 125.1 | ×75.9 | 61.0 | ×114.5 | 99.7 | ×100.2 | ×96.9 ± 15.9 |
| Pm7 | 1 | 9498.1 | 6986.5 | 9988.6 | ||||
| Pm7 + L-NMMA | 21 | 113.6 | ×74.9 | 73.7 | ×114.2 | 62.8 | ×122.0 | ×103.7 ± 20.6 |
| Pm7 + L-NMMA | 1 | 8504.7 | 8413.8 | 7660.6 | ||||
| Pm7 + hGATA-2 | 21 | 118.1 | ×138.8 | 112.0 | ×171.2 | 61.0 | ×132.0 | ×147.3 ± 17.1 |
| Pm7 + hGATA-2 | 1 | 16386.4 | 19175.9 | 8050.9 | ||||
| Pm7 + hGATA-2 + L-NMMA | 21 | 182.7 | ×124.6 | 111.5 | ×135.8 | 126.1 | ×110.0 | ×123.5 ±10.6 |
| Pm7 + hGATA-2 + L-NMMA | 1 | 22766.1 | 15145.5 | 13870.4 | ||||
| . | % O2 . | Experiment 1 . | Experiment 2 . | Experiment 3 . | Fold induction (mean ± 1 SD) . | |||
|---|---|---|---|---|---|---|---|---|
| RLU/CAT . | Fold induction . | RLU/CAT . | Fold induction . | RLU/CAT . | Fold induction . | |||
| Pwt | 21 | 56.6 | ×50.6 | 75.8 | ×63.4 | 54.3 | ×53.5 | ×55.8 ± 5.5 |
| Pwt | 1 | 2866.4 | 4807.9 | 2902.8 | ||||
| Pwt + L-NMMA | 21 | 42.6 | ×38.0 | 72.9 | ×41.0 | 46.1 | ×20.1 | ×33.0 ± 9.2 |
| Pwt + L-NMMA | 1 | 1619.8 | 2985.7 | 927.0 | ||||
| Pwt + hGATA-2 | 21 | 50.7 | ×24.8 | 80.4 | ×23.4 | 49.4 | ×16.2 | ×21.5 ± 3.8 |
| Pwt + hGATA-2 | 1 | 1257.1 | 1877.8 | 799.8 | ||||
| Pwt + hGATA-2 + L-NMMA | 21 | 57.7 | ×19.1 | 96.3 | ×11.7 | 45.6 | ×11.9 | ×14.2 ± 3.4 |
| Pwt + hGATA-2 + L-NMMA | 1 | 1101.6 | 1130.2 | 544.2 | ||||
| Pm7 | 21 | 125.1 | ×75.9 | 61.0 | ×114.5 | 99.7 | ×100.2 | ×96.9 ± 15.9 |
| Pm7 | 1 | 9498.1 | 6986.5 | 9988.6 | ||||
| Pm7 + L-NMMA | 21 | 113.6 | ×74.9 | 73.7 | ×114.2 | 62.8 | ×122.0 | ×103.7 ± 20.6 |
| Pm7 + L-NMMA | 1 | 8504.7 | 8413.8 | 7660.6 | ||||
| Pm7 + hGATA-2 | 21 | 118.1 | ×138.8 | 112.0 | ×171.2 | 61.0 | ×132.0 | ×147.3 ± 17.1 |
| Pm7 + hGATA-2 | 1 | 16386.4 | 19175.9 | 8050.9 | ||||
| Pm7 + hGATA-2 + L-NMMA | 21 | 182.7 | ×124.6 | 111.5 | ×135.8 | 126.1 | ×110.0 | ×123.5 ±10.6 |
| Pm7 + hGATA-2 + L-NMMA | 1 | 22766.1 | 15145.5 | 13870.4 | ||||
Wild-type (Pwt) and mutated (Pm7) GATA motifs. Hypoxic induction of the Luc gene expression is represented as a hypoxia/normoxia ratio. Fold induction indicates this hypoxia/normoxia ratio. Three separate experiments (duplicate sample) were performed (n = 3).