Figure 3

p53 can bind chromatin region surrounding the identified p53 REs in miR genes. A) ChIP assays were performed in HCT116 p53^+/+(gray bars) and HCT116 p53^−/− (black bars) upon doxorubicin treatment for 24 hours. The results of Real-Time qPCR are presented as fold of mock treatment, normalized with respect to the signal obtained with Input DNA. The results from three control locations corresponding to promoter regions of Actin, GAPDH and exon 9 of CCNB1 genes were averaged and are also presented in panel A (Neg. Ctrls). P21 and miR-34a occupancy were measured as positive controls. Bars represent average and standard deviations of three independent experiments. C) ChIP assays of MCF7 cells treated with doxorubicin for 24 hours. Results obtained after ChIP with an antibody against IgG were included as a negative control for the p53 miR-REs. Examples of agarose gel analysis of standard ChIP-PCR are given in panels B and D. Specifically, panel B shows experiments performed in HCT116 p53^+/+ and p53^−/− cells, while panel D presents results from MCF7 cells. The DO-1 p53 antibody was used for immunoprecipitation; NTC, no template control. Regions surrounding the established P21 and miR-34a p53 REs were examined as positive controls.