Figure 3.

EGFP fluorescence in HT 1080 human fibrosarcoma cells transfected with the hypoxia-responsive 5HRE-hCMVmp-d2EGFP vector (destabilized EGFP protein, half-life 2 hours), after treatment at oxygen concentrations between 20% and <0.02% for 1 hour (A), 4 hours (B) and 12 hours (C), respectively, with or without a 4-hour reoxygenation period. Fluorescence is given as the percentage of fluorescence in positive control cells transfected with CMV-d2EGFP vector (mean±SEM, n= 4). Western blot of EGFP protein in cells transfected with 5HRE-hCMVmp-d2EGPF vector (5HRE), immediately after 12 hours at different O₂ concentrations, with aerobic CMV-transfected and wild-type (WT) cells for comparison (D). At <0.02% O₂, the EGFP protein level in 5HRE-hCMVmp-d2EGFP-transfected cells is lower than at 0.06% or 0.2%, suggesting reduced EGFP protein synthesis under near-anoxic conditions, and not only unoxidated chromophore, as an explanation for the drop of fluorescence at the lowest oxygen concentration in (C).