Figure 4

I3C induces p53-dependent apoptosis and regulation of MDM2–p53 and MDM2–nucleostemin protein–protein interactions. (A) 10AT-Her2 and 10AT-Neo cells were treated with or without 200 μM I3C for the indicated durations and cell number was quantified by the cell proliferation assay described in the Methods section. (B) 10AT-Neo and 10AT-Her2 cells were treated with or without 200 μM I3C for 48 hours. The DNA content of nuclear DNA stained with propidium iodide was assessed by flow cytometry. (C) 10AT-Her2 cells were treated with or without 200 μM I3C for 48 hours, total cell extracts were electrophoretically fractionated and then Western blots probed for PARP, Akt1, p53 and the actin gel loading control. (D) 10AT-Her2 cells were transfected with either a dominant negative p53 (DN p53) expression vector or with the empty expression vector (EV), and then treated with or without 200 μM I3C for 48 hours. The DNA content of nuclear DNA stained with propidium iodide was assessed by flow cytometry. (E, F) 10AT-Her2 cells were treated with or without 200 μM I3C for 48 hours. Total cell extracts were immunoprecipitated with either MDM2 (E) or nucleostemin (F) antibodies. As a control, non-immune antibodies (of immunoglobulin G or IgG) and samples not immunoprecipitated (No IP) were used. All extracts were electrophoretically fractionated and probed by Western blot analysis using antibodies specific to p53, serine-166 phosphorylated MDM2 or total MDM2 (E) or with antibodies specific to either serine-166 phosphorylated MDM2 or total MDM2 (F). DN, dominant negative; EV, empty expression vector; I3C, indole-3-carbinol; IgG, immunoglobulin G; IP, immunoprecipitated; MDM2, murine double mutant 2; PARP, poly ADP ribose polymerase.