Figure 1. Effects of hypoxia and reoxygenation on NF-κB activation.

A. Representative EMSAs for measurement of NF-κB activity under normoxic (21% 0₂) and hypoxic (1% 0₂) conditions for the indicated times. WT and M = cold-competition with 100-fold molar excess of wild-type and mutant NF-κB DNA response elements, respectively. Red = HPV-positive squamous cell carcinoma; blue = HPV-negative squamous cell carcinoma; black = non-squamous cell carcinoma. B. NF-κB reporter assays performed 48 hrs after transfection. N = normoxia; H = hypoxia. Results are averages of three experiments ± standard deviation (s.d.). Firefly luciferase expression was normalized to that of Renilla luciferase (see Methods). C. Electrophoretic mobility supershift assays were performed with the indicated antibodies after cells were exposed to hypoxia for 24 hours. D. Top panels: EMSAs demonstrate detailed time course of hypoxia-induced NF-κB activity. Second row: Oct-1 EMSAs serves as a control. Third row: HIF1α Western blots confirm exposure to hypoxia. Bottom row: γ-tubulin Westerns as loading controls. E. Effects of reoxygenation on NF-κB activity (measured by EMSA). EMSAs for AP1 or Oct-1 served as controls. Cells were exposed to hypoxia for 24 hrs followed by return to normoxic conditions (reoxygenation) for the indicated times. F. Transient hypoxia-induced NF-κB activation in HPV-negative cancer cells. Top panels: Shorter course hypoxia exposure (1 hr) of HPV-negative cancer cells. Bottom panels: detailed time course of transient NF-κB activation.