Effects of IFN-γ on heparanase expression in endothelial cells. (A) Semiquantitative RT-PCR. EA.hy926 cells were incubated (16 hours) in triplicate in the absence or presence of 80 ng/mL IFN-γ. RNA was then isolated from the cells, and comparative semiquantitative PCR was performed as described in “Materials and methods.” Aliquots (10 μL) of the PCR products were separated by 1.5% agarose gel electrophoresis and visualized (top). The intensity of each band was quantitated using Scion Image software, and the results are expressed as band intensity relative to that of the housekeeping gene L19. The bars represent the mean ± SD (error bars) of 3 independent experiments (bottom). (B) Heparanase activity. EA.hy926 cells were incubated (16 hours) in the absence (□), or presence (♦) of 80 ng/mL IFN-γ. Cell lysates were normalized for equal protein and incubated (3 hours, pH 6.0, 37°C) with sulfate-labeled ECM. Labeled degradation fragments released into the incubation medium were analyzed by gel filtration on sepharose 6B.