Reporter assays

To construct the EPO enhancer-driven luciferase reporter gene, the human EPO enhancer region (5-GGTACCGGCCCTACGTGCTGTCTCACACAGCCT GTCTGACCTCTCGACCTACCGGCCAGATCT-3) was inserted into the multi-cloning site of the pGL4 vector (Promega, Madison, WI). Mutated EPO luciferase was generated by altering the DNA sequence targeted by HIF-1 (CGTG to AAAA). Hep3B cells were transfected with 1 mg of the reporter plasmid using Lipofectamin 2000 (Life Technologies). After stabilized for 48 hours, the Hep3B cell lines stably harboring the reporter plasmid were selected under G418, and five stable cell lines were mixed to rule out the artifact generated by the integration of the plasmid into chromosomes. The cell line was pre-treated with each compound in a chemical library for 4 hours, and then incubated under normoxia or hypoxia for 16 hours. Cells were lysed and luciferase activity was measured using a LB960 luminometer (Berthold Technologies, Oak Ridge, TN). To evaluate the cap-dependent and the IRES-dependent translation of HIF-1α mRNA, we used thymidine kinase (TK) promoter/HIF-1α 5′-UTR/luciferase and CMV promoter/GFP/HIF-1α 5′-UTR/luciferase reporters, respectively [41]. The β-gal expression plasmid was cotransfected into cells to normalize transfection efficiency.