Figure 2.

BC005927 is induced by hypoxia inducible factor‐1 alpha (HIF‐1α), and HIF‐1α interacts with the HIF‐1α response element in the BC005927 promoter region. A, Schematic illustration of consensus HIF‐1α response element in BC005927 gene promoter. B, C1, C2, SGC7901, MKN45 cells expressing control shRNA, HIF‐1α shRNAs were cultured under hypoxic conditions for 24 hours. Cell lysates and total RNA were subjected to western blot and real‐time RT‐PCR analyses, respectively. Data shown are mean ± SD (n = 3). D1, D2, Cell lysates and total RNA of MKN28 cells expressing NC (negative control) or pc‐DNA‐HIF‐1α were subjected to western blot and real‐time RT‐PCR analyses, respectively. Data shown are mean ± SD (n = 3). E, SGC7901 cells were cultured under normoxic or hypoxic conditions for 24 hours. Lysates were then subjected to ChIP assay. ChIP products were amplified by PCR reaction. F1, F2, SGC7901 cells were cotransfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty‐four hours after transfection, cells were cultured under normoxic or hypoxic conditions for 24 hours. Reporter activity was then measured and plotted after normalizing with respect to Renilla luciferase activity (mean ± SD). G1, G2, SGC7901 cells expressing control shRNA, HIF‐1α‐si1, or HIF‐1α‐si2 were cotransfected with the hypoxia response element (HRE)‐WT reporter constructs and Renilla luciferase plasmid. Twenty‐four hours after transfection, cells were cultured under normoxic or hypoxic conditions for 24 hours. Reporter activity was then measured and plotted after normalizing with respect to Renilla luciferase activity (mean ± SD). In all panels, results are representative of at least 3 independent experiments. **P < .01