Figure 3. Hypoxic dual luciferase expression in ARPE-19 cells transfected with the Rpe65 promoter, the HRSE-Rpe65 promoter containing three hypoxia-regulated elements, and the HRSE-6xHRE-Rpe65 promoter. The addition of the three hypoxia-regulated elements (HREs) in HRSE-Rpe65 accounts for a modest increase in basal Rpe65 inducibility (black). Transiently transfected cell lines were exposed to forty hours of hypoxia (black) or normoxia (white). All promoter activity was normalized to the aerobic activity of the Rpe65 promoter. A small hypoxic induction of 1.6±0.3 (n=7, p>0.05) fold was identified in the native Rpe65 promoter. Ligation of an upstream HRSE to the Rpe65 promoter resulted in a 9.4±5.8 (n=9, p<0.001) fold hypoxic induction due to three HREs present in the HRSE. Addition of the 6xHRE oligomer brings the total number of HREs to nine and accounts for the 51.3±10.8 (n=10, p<0.001) fold increase in hypoxia inducibility over aerobic baseline. Asterisks (*) indicate statistical significance.