Figure 2

p53 and Mdm2 nuclear localization and chromatin association are not influenced by estrogen treatment. MCF-7 cells were treated with 10 nM estrogen (E2) for five days and 50 μM etoposide (ETOP) for 48 hours. (a) Immunofluorescence was carried out for p53 and Mdm2 proteins. Nuclear DNA was stained with DAPI. 400 μg of cross-linked and sonicated whole cell lysates were subjected to chromatin immunoprecipitation (ChIP), using antibodies against p53 (b) or Mdm2 (c). ChIP with non-specific IgG was done to subtract the background. Immunoprecipitated DNA was amplified by real-time quantitative PCR with primers and FAM-labeled probes for p53-REs in puma, mdm2 and p21 genes. Values were normalized to IgG and inputs, followed by normalization to the DSMO samples. Graphs show means and standard errors of three independent experiments. The changes in relative binding of p53 and Mdm2 to the three different p53-REs were not significant (P > 0.05). The P-values calculated by student T-test for the difference between with or without E2 were all between 0.1 and 0.25.