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. 2007 Aug 13;406(Pt 2):317–323. doi: 10.1042/BJ20070286

Figure 4. Tax induces HRE reporter activity through the PI3K/Akt signalling pathway.

Figure 4

(A) HeLa cells were transfected with the following expression vectors: increasing amounts (0, 0.5, 1 or 2 μg) of Tax WT, HIF-1α or a combination of 0.5 μg of Tax WT and increasing amounts (0, 0.5, 1 or 2 μg) of HIF-1α, together with 0.5 μg of luciferase reporter plasmid containing the HRE (pGL3-6×HRE-Luc) as indicated. After 48 h, cells were collected for the luciferase assay. (B) Tax induced HIF-1α expression in HeLa cells. HeLa cells were transfected with increasing amounts (0, 1, 2 or 4 μg) of Tax WT. Cells were harvested 48 h after transfection and analysed for HIF-1α and Tax expression by Western blotting. (C) A combination of Tax and HIF-1α transfection caused a more pronounced accumulation of HIF-1α. HeLa cells were transfected with 0.5 μg of Tax WT and 2 μg of HIF-1α as indicated in the Figure. Cells were harvested 48 h after transfection and analysed for HIF-1α and Tax expression by Western blotting. (D) HeLa cells were transfected with the following expression vectors: increasing amounts (0, 0.5, 1 or 2 μg) of Tax WT or Tax mutants (M22, 703 or K88A), together with 0.5 μg of luciferase reporter plasmid containing the HRE (pGL3-6×HRE-Luc) as indicated. After 48 h, cells were collected for the luciferase assay. (E) Tax-enhanced HRE reporter activity was inhibited by a PI3K inhibitor. HeLa cells were transfected with 2 μg of HIF-1α and 0.5 μg of Tax WT or mutant (M22, 703 or K88A) expression plasmids, together with 0.5 μg of pGL3-6×HRE-Luc as indicated in the Figure. After 24 h, cells were treated with increasing amounts of LY294002 (0, 5, 10 or 20 μM) for a further 24 h. (F) Tax-enhanced HIF-1 reporter activity was inhibited by AKT-DN. HeLa cells were transfected with 2 μg of Tax WT, or a combination of 0.5 μg of Tax WT and 2 μg of HIF-1α as indicated in the Figure, together with increasing amounts (0, 0.05, 0.1 or 0.5 μg) of AKT-DN and 0.5 μg of pGL3-6×HRE-Luc. After 48 h, cells were collected, and transcriptional activity was determined using the luciferase assay. Relative luciferase activities were measured in cell extracts normalized to the Renilla luciferase activity. Luciferase activity is presented as a fold induction relative to the basal level measured in cells transfected with the reporter plasmid alone. Values are the means±S.D. from three separate experiments.