Figure 3

HIF-1α binds to HRE within MDR-1 and VEGF gene promoters in MCF7 3-D spheroids. The binding of HIF-1α on MDR-1 and VEGF gene promoters were measured by ChIP assay as described in Materials and Methods. A: PCR analysis for the MDR-1 gene promoter region containing a HRE was performed on immunoprecipitation samples (IP) with anti-HIF-1α antibody and with purified total input DNA (input DNA) from MCF7 control cells (Monolayer Normoxia); MCF7 hypoxic cells (Monolayer Hypoxia); and MCF7 3-D spheroids (Spheroids). B: PCR analysis for the VEGF gene promoter (an established HIF-1 target gene) was performed on immunoprecipitation samples with anti-HIF-1α antibody and with purified total input DNA from MCF7 control cells; MCF7 hypoxic cells; and MCF7 3-D spheroids. C. PCR analysis for the MDR-1 gene promoter region that does not contain a HRE was performed on immunoprecipitation samples (IP) with anti-HIF-1α antibody and with purified total input DNA from MCF7 control cells; MCF7 hypoxic cells; and MCF7 3-D spheroids. Cell monolayers and spheroids were incubated in the absence (-) or in the presence (+) of 5 μM YC-1 for 24 h. The figures are representative of three similar experiments.