FIGURE 1.

PTEN regulates expression of HIF1α protein. A, muristerone-inducible PTEN or G129E and G129R mutants of PTEN engineered in the U87MG cell lines were used to detect the expression of PTEN and pAKT before and after induction of 0.5 μmol/liter muristerone. After 36 h of muristerone induction, cell lysates were prepared and used for Western blot analysis of PTEN and pAKT. B–D, muristerone induced expression of PTEN in the U87 cell line including wild type PTEN orG129E and G129R PTEN mutant expression. B–D, GFAP V12 Ras PTEN^fl/fl (B) or GFAP V12 Ras PTEN^fl/fl cre cell lines (C) and MEF PTEN^+/+ and MEF PTEN^−/− cell lines (D) were serum-starved for 4 h and were kept in normoxic (21% O₂) or hypoxic (1% O₂) for 4 h followed by preparation of nuclear extracts (for HIF1α) as well as WCE and Western blot analysis. E, MEF PTEN^−/− cells were transfected with 10 μg of empty vector or HA-PTEN plasmid using Lipofectamine Plus. 48 h after transfection, cells were serum-starved for 4 h followed by hypoxia (1% O₂) for 4 h. Cells were used for preparation of nuclear extracts (for HIF1α) and whole cell extracts for HA and pAKT for Western blot. Cell lysates were run on SDS-PAGE, and proteins were transferred on nitrocellulose membrane and probed with HIF1α, HA or pAKT (Ser⁴⁷³) antibodies. F, LN229-HRE-AP cell lines were transfected with different concentrations of HA-PTEN (6, 8, and 10 μg), C124S (10 μg) mutant, and control plasmid (10 μg) using FuGENE transfection reagent. After 48 h of transfection, cells were serum-starved for 4 h and exposed to hypoxia (1% O₂) for 4 h followed by preparation of nuclear extracts and whole cell extracts for Western blot analysis. G, LN229-HRE-AP cell lines stably transfected with PTEN shRNA or a control construct as described under “Experimental Procedures” were serum-starved for 4 h followed by hypoxia (1% O₂) for 4 h and preparation of nuclear extracts and Western blot analysis. The experiments were repeated three times with similar results.