FIGURE 2.
PTEN regulates HIF1α-dependent target gene expression. A, mRNA was isolated from GFAP V12 RasPTEN f/fl or GFAP V12 Ras PTENfl/fl cre cell lines and the expression of VEGF, HK1, PGK-1, and GLUT1 probes were analyzed by real time PCR. B, different concentrations of HA-PTEN plasmid vector (6, 8, and 10 μg) DNA or its mutant C124S (10 μg) were transfected into serum-starved LN229-HRE-AP cells. After 48 h of transfection, cells were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 h, followed by preparation of lysates, and HRE-mediated AP activity was read at 405 nm using p-nitrophenyl phosphate as a substrate. Cells transfected with pcDNA were used as a control. C, LN229-HRE-AP cell lines stably transfected with PTEN shRNA or a control construct were subjected to normoxia (21% O2) or hypoxia (1% O2) for 24 h, and HRE-mediated AP activity was read at 405 nm. D, RNA was isolated form LN229-HRE-AP cell lines stably transfected with PTEN shRNA and its control constructs in normoxic (21% O2) and hypoxic (1% O2) conditions and used to quantitate expression of VEGF, HK1, PGK-1, and GLUT1 genes by real time PCR. The data are representative of two or three independent experiments. Graphs present means ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus GFAP V12 Ras PTENfl/fl (A), vector (B), and control shRNA (C and D) in hypoxic conditions as determined using pair wise two-sided Student's t test. Please note that the expression values of all genes (A and D) and alkaline phosphatase activity (B and C) in hypoxic conditions are significant compared with normoxic conditions.