p53 binds 14-3-3γ promoter and negatively regulates 14-3-3γ expression. A. Yeast one hybrid assays. The 600 and 1200 bp 14-3-3γ promoter sequences were subcloned into the GFP reporter vector as described in materials and methods. The p53 encoding plasmid was co-transformed with reporter vectors into Saccharomyces cerevisiae strain W303 and successful transformants were used for the GFP fluorescent assay in the presence of UV. The results were normalized to cells grown in the sucrose medium. Values in y-axis are represented as fold change. Experiments were repeated three times and p values calculated using student's t-test (*p < 0.05). B. A schematic diagram of the 1200 bp of the human 14-3-3γ gene promoter is shown with the position and sequence of the putative p53 binding motif identified. The p53 consensus binding motif is shown for comparison. Asterisks denote identity between the two sequences. This promoter fragment was cloned into PGL3 adjacent to the luciferase gene (shown at right). C. CHIP assays in lung cancer cell lines. A549, H358 and H322 cells were either left untreated (A549, H358, H322) or exposed to 10 Gy ionizing radiation (A549-IR, H358-IR, H322-IR). The cells were harvested eight hours after γ-irradiation and chromatin immunoprecipitation performed as described in the methods section. Reactions were performed either with a mouse IgG as a negative control (A549 -p53ab, H358 -p53ab, H322 -p53ab) or the anti-p53 DO1 antibody. PCR products were separated on agarose gels and visualized by staining with ethidium bromide and viewing with UV illumination. Three independent experiments were performed. Typical results are shown. D. CHIP assays in human colon cancer cell lines. HCT116p53+/+ or HCT116p53-/- cells were either left untreated or exposed to 10 Gy ionizing radiation and chromatin immunoprecipitation reactions performed as in panel C using either a mouse IgG as a negative control (-p53ab) or with the anti-p53 DO1 antibody. PCR products were visualized as in panel C. Reactions using input DNA are shown at the bottom of the panel. Three independent experiments were performed. A typical result is shown. E. 14-3-3γ promoter reporter assays in lung cancer cell lines following γ-irradiation. A549, H322, or H358 cells were transiently transfected either with empty vector (open bars) or with the reporter depicted in panel B (solid bars). Forty eight hours after transfection the cells were either left untreated (A549, H358, H322) or exposed to ionizing radiation (10 Gy). The cells were harvested eight hours after gamma-irradiation and luciferase activity measured and normalized relative to renilla luciferase to control for variations in transfection efficiency. The bars represent the average of three experiments ± SD. An asterisk denotes those values that are significantly reduced relative to unirradiated controls (*p < 0.01; control vs IR). F. 14-3-3γ promoter reporter assays in colon cancer cell lines following γ-irradiation. HCT116p53+/+ or HCT116p53-/- cells were treated in the same way as cells in panel E. Luciferase activity was determined in the non-irradiated and irradiated (IR) cells as described above. The bars depict the average of three independent experiments, ± SD. Open bars show values for cells transfected with the empty vector. Solid bars show values for cells transfected with the reporter vector shown in panel B. An asterisk marks those values that were significantly reduced relative to non-irradiated controls (*p < 0.01; control vs IR).