Fig. 4. The effect of PI3K activity on HIF-1{alpha} mRNA levels. PC-3 and DU145 cells were cultured in serum-free medium for 24 h. The PI3K inhibitor LY294002 was added at the indicated concentration 30 min before the addition of insulin and EGF, followed by incubation in the absence (-) or presence (+) of 200 nM insulin or 20 ng/ml EGF as indicated for 6 h. Total RNA was isolated, and 10-µg aliquots were separated by electrophoresis in 2.2 M formaldehyde/1.1% agarose gels and transferred to a nylon membrane. The membrane was probed by using a 32P-labeled HIF-1{alpha} cDNA fragment.