Zymographic analysis of PAs present in the medium of HTR-8/SVneo cell cultures incubated for 8, 12, and 24 hours under normoxic or hypoxic conditions. Casein and plasminogen were incorporated into the acrylamide before polymerization as detailed elsewhere.17 Samples of serum-free medium containing 200 ng of protein were loaded onto each lane and separated by electrophoresis. After an overnight incubation in 5 mmol/L CaCl₂ in TRIS buffer, gels were stained with Coomassie R-250 in 10% acetic acid/40% methanol and destained in 10% acetic acid/40% methanol. Clear areas represent caseinolytic activity. Note caseinolytic bands at 50 to 55 kD corresponding in size to uPA, and that at 24 hours of hypoxic culture, the caseinolytic bands were not discernible.