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. 2017 Feb 27;7(5):1100–1113. doi: 10.7150/thno.18198

Figure 6.

Figure 6

HIF1α can directly binds to the AHNAK2 promoter. (A) Simplified schematic showing the putative hypoxia-response elements and truncated sites in the human AHNAK2 promoter region, +1 indicates translational start site. 293T cells were transfected with the AHNAK2-pGL4.17-firefly reporter construct and a control Renilla expression vector. Luciferase assay was performed under hypoxic conditions, HIF1α over-expression (B) and CoCl2 treatment (200 μM, treated for 48 hours) (C), * p < 0.05, ** p < 0.01, *** p < 0.001. (D) PCR amplification of DNA fragments immuno-precipitated by anti-HIF1α. CAKI-1 cells were cultured under hypoxia conditions for 24 hours and subjected to ChIP assay. (E) The control Renilla expression vector was co-transfected with the AHNAK2-pGL4.17 luciferase reporter construct (AP20) or truncated reporter plasmid (AP18, AP16, AP12) into 293T cells. Luciferase activity assay was performed 48 hours after transfection. * p < 0.05. (F) Luciferase activity of mutant AHNAK2 promoter reporters in CAKI-1 cells normalized to wild type. The pGL4.17-basic luciferase construct was used as a negative control. Three independent experiments were performed in duplicate. Each bar represents mean ± SD. *** p < 0.001.