Fig. 3

HIF1α regulates the expression of PDGFRA and PDGFD in GBM cells. A WT U251 cells were cultured in the indicated hypoxic conditions and protein expression was detected by Western blot. B Co-expression of HIF1α with PDGFRα and PDGF-D in GBM tissue arrays were examined by immunofluorescence co-staining. C-F. HIF1α directly regulates PDGFRA promoter activity. C Diagram of 5′ region of the PDGFRA gene, with positions of three HRE sites, its proximal promoter region, intronic enhancer regions and locations of ChIP primers, is shown. D Induction of PDGFRA promoter activity by HIF1α. HEK293 cells were co-transfected with plasmids of PDGFRA promoter and either empty vector control or HIF1α-PPN. Twenty-four hours after transfection, GFP expression was visualized by fluorescence microscopy, depicted in the representative photographs. PDGFRA-P1 corresponds to the promoter construct containing promoter alone; E1, E2 denote either of the intronic enhancers. E GFP mean fluorescence intensity (MFI) was quantitated by flow cytometry and summarized for cells transfected in triplicate. F HIF1α binds to the enhancers of PDGFRA promoter. ChIP assay was conducted in U251 cells using rabbit HIF1α antibody and rabbit IgG as a parallel control. One tenth of lysate was used as the input and the data shown is representative of 3 experiments. G, H Induction of PDGFD promoter activity by HIF1α was performed as in D,E. I HIF1α binds to the proximal HRE site of the PDGFD promoter as in F. The SD is from three independent experiments each performed in triplicate