Figure 2.

Analysis of the human DNA-PKcs promoter in HeLa and MCF-7 Cells. (A) DNA-PKcs promoter region showing putative binding sites for ERα, Sp1 and a potential initiator consensus element (Inr). Differences to the core 13 bp consensus ERE are labelled in red. (B) HeLa cells were transfected with the DNA-PKcs promoter luciferase reporter plasmid and ERα expression plasmid and treated with 100 nM of E2 for 24 h. The luciferase activities were normalized to the internal transfection control and values obtained from cells receiving vehicle (−) were set as 1. Empty pGL3-promoter vector was used as a negative control. Data represent the mean of three independent experiments performed in triplicate. ^*P<0.05. (C) MCF-7 cells were transfected with DNA-PKcs promoter luciferase reporter plasmids and treated with 100 nM of E2 for 24 h. The luciferase activities were normalized to the internal transfection control, and values obtained from cells receiving vehicle (−) were set as 1. Data represent the mean of three independent experiments performed in triplicate. ^*P<0.05. (D) Cells were grown in phenol red-free medium supplemented with 10% charcoal–dextran-stripped FBS for 3 days. After treatment with 100 nM of E2 for 1 h, cells were crosslinked with 1% formaldehyde and monitored by ChIP assays. Soluble chromatin from control and E2-treated MCF-7 cells was immunoprecipitated with anti-ERα or a control IgG. The final DNA extractions were amplified by PCR using pairs of primers that cover the indicated EREs or CR of the DNA-PKcs promoters. ChIP, chromatin immunoprecipitation; CR, control region; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; E2, oestrogen; ERα, oestrogen receptor-α; ERE, oestrogen-responsive element; FBS, fetal bovine serum; ICI, ICI 182,780; IgG, immunoglobulin G; IP, immunoprecipitation.