LPS stimulates HIF-1 transcriptional activity. (A) NR8383 cells (5 × 105 cells/well, 6-well plate) were transfected with 500 ng PRE-tk-LUC reporter construct and 250 ng of an expression vector coding for Renilla reniformis luciferase was used to normalize for transfection efficiency. (B) NR8383 cells were cotransfected with luciferase vectors as in panel A and either 4 μg pcDNA3 (□)or 4 μg pcDNA3-HA-DN-HIF-1α (▦). At 12 hours after transfection, cells were deprived of FBS for 16 hours. Cells were then maintained under either control conditions, hypoxic conditions, or in the presence of LPS (1 μg/mL) for 18 hours. At this point, NR8383 cells were lysed and luciferase activity was measured using the Dual-Luciferase Reporter Assay System. Results are expressed as a ratio of beetle luciferase activity over Renilla reniformis luciferase activity. Data expressed are an average ± SD of at least 3 independent experiments performed in triplicate.
