Figure 4

VHL knockdown and the condition of hypoxia induce activation of HIF-1 transcription and transactivation of the CXCR4 promoter. (A). SN12C-VC and SN12C-VHL-KD cells were serum-starved and exposed to either normoxia or hypoxia for the times indicated. Subsequently, nuclear extracts were prepared and analyzed by SDS-PAGE and Western blotting with a specific mouse-anti-human HIF-1α monoclonal antibody. (B). SN12C-VC and SN12C-VHL-KD cells were transfected with a reporter construct comprising a 2.6-kilobase fragment of the CXCR4 promoter and a separate Renilla control reporter. Cells were co-transfected with either a control vector expressing GFP or a vector expressing HIF-1α. The transfected cells were subsequently exposed to either normoxia or hypoxia for 8 hours. Cell extracts were prepared and analyzed in a luminometer. Relative light units (RLU) were normalized to the Renilla control. (C). SN12C-VC and SN12C-VHL-KD cells were serum-starved and exposed to either normoxia or hypoxia for 4 h. ChIP assay was subsequently performed to investigate the recruitment of HIF-1α on the CXCR4 promoter. Precleared chromatin solutions were immunoprecipitated with either mouse anti-human HIF-1α monoclonal antibody or with no antibody as negative controls (N). The lower panel represents input genomic DNA before addition of antibody.