Figure 3. Hypoxia, EC-specific growth medium, and nutrient deprivation induce Tie2-dependent EGFP expression in tumor cells.

Separate cultures of H460 and OVCAR3 tumor cells were stably co-transfected with Tie2-EGFP plasmid and pFR400 encoding a mutant form of dihydrofolate reductase [36,37] and selected in G-418 and increasing concentrations of methotrexate to allow amplification of the two tandemly integrated vectors and a corresponding increase EGFP signal intensity. Cells were exposed to conditions shown previously to induce the maximal TDEC phenotype (i.e., condition 3 for H460’s and condition 4 for OVCAR3) and compared to control cells grown under standard conditions. (A) Fluorescence micrographs of each cell line after five days of growth under each set of conditions. (B) Flow cytometric analysis of the same cells. Representative results of at least three independent experiments are depicted. Scale bar = 25 um.