An analysis of HIF-1α expression through immunocytochemistry, protein expression, and functional assays. (A) Immunocytochemical analysis of celluar HIF-1α revealed a qualitative increase in nuclear HIF-1α presence in cells exposed to either hypoxia or 5 Gy radiation over normoxic and nonradiated controls, respectively. A combination of hypoxia and 5 Gy radiation dose showed a greater qualitative luminosity of HIF-1α staining over both hypoxia and radiation-exposed samples individually. (B) Quantification of in vitro HIF-1α luminosity by photometric analysis revealed a statistically significant increase in HIF-1α nuclear localization in hypoxic and radiated cells versus normoxic nonradiated controls. A combination of hypoxia and 5 Gy radiation revealed a statistically significant increase in HIF-1α nuclear localization over hypoxia or radiation alone (n = 6). (C) HIF-1α protein was assessed by Western blot at both 48 and 72 hours ± normoxia, hypoxia, or 5 Gy IR. Radiation was a strong stimulus for HIF-1α in both hypoxic and normoxic conditions. The strongest response was seen when hypoxia and radiation were combined. There is also increased cytoplasmic stabilization of HIF-1α starting at 48 hours. (D) Functional cellular activity of HIF-1α was determined through a luciferase reporter construct containing a HRE on the promoter. Statistically significant increases in luciferase activity were seen after 5 Gy IR in both hypoxia and normoxia (n = 6).