FIGURE 3.

Inhibition of PI3K pathway blocks hypoxic accumulation of HIF1α. A, serum-starved LN229-HRE-AP cells (4 h) were treated with 250 nm, 500 nm, and 1 μm PF4691502 for 30 min followed by IGF stimulation and then placed under normoxic or hypoxic conditions (1% O₂) for 4 h followed by the preparation of nuclear extracts for Western blot analysis of HIF1α and WCE for pAKT and AKT blots. B and C, serum-starved U87 and LN229-HRE-AP cells treated with 25 μm SF1126, 500 nm PF4691502, BEZ, and BKM120 inhibit HIF1α accumulation under hypoxia in U87 (B), LN229-HRE-AP (C) cells. Cells were serum-starved for 4 h pretreated with inhibitors for 30 min followed by IGF stimulation and then placed under normoxic or hypoxic conditions (1% O₂) for 4 h followed by the preparation of nuclear and whole cell extracts for Western blot analysis. D, serum-starved LN229-HRE-AP cells were pretreated with 100 nm GDC0941 and TGX221 for 30 min followed by IGF stimulation and then placed under normoxic or hypoxic conditions (1% O₂) for 4 h followed by the preparation of nuclear extracts for Western blot analysis of HIF1α and WCE for pAKT and AKT blots. E, MEF PTEN^+/+ cells were transfected with 10 μg of Myr-AKT or empty vector using Lipofectamine Plus transfection reagent, and 48 h after transfection, cells were serum-starved for 4 h and subjected to hypoxia for 4 h followed by preparation of nuclear extracts and whole cell extracts. F, LN229-HRE-AP cells were transfected with 10 μg of Myr-AKT or empty vector using FuGENE transfection reagent and 48 h after transfection. The cells were serum-starved for 4 h pretreated with 500 nm of PF4691502 for 30 min followed by IGF stimulation and then placed under hypoxic conditions (1% O₂) for 4 h followed by the preparation of nuclear extracts and WCE for Western blot analysis. The data are representative of two or three independent experiments.