Screening system for demethylation agents. (A) Schematic depiction of the reporter system in MCF7 cells. The reporter gene system consists of two constructs (top). Expression of Tet repressor (TetR) was under the regulation of Trip10 promoter. The addition of doxycycline (Dox) or targeted methylation of Trip10 promoter (center) blocks the silencing/binding of Tet repressor onto the Tet operator (TetO2) thus the EGFP would express and become detectable with fluorescence microscope. Bottom, demethylation of the Trip10 promoter restores the expression of Tet repressor and thus silences the EGFP. Open circles: unmethylated CpG dinucleotides; filled circles: methylated CpG dinucleotides. (B) Validation of the reporter gene system. As shown, presence of doxycycline did not affect the cell density, but increased the intensity of EGFP substantially (top). Histograms show the quantified EGFP intensity in the presence or absence of doxycycline (bottom). The EGFP intensity was analyzed and quantified by Image J. Columns, mean (n = 3); error bars, S.D. (C) Tracking of the transfected me_Trip10 DNAs. In vitro methylated Trip10 promoter DNAs were labelled with or without (control) Cy5 and then used to transfect the MCF7 cells. There is a high degree of overlap between the EGFP-expressing cells and Cy5-positive cells, suggesting the high transfection efficiency in this system.