Figure 7
Effects of nucleostemin knockdown on I3C-induced apoptotic response and regulated MDM2- p53 protein interactions. (A) 10AT-Her2 cells transfected with nucleostemin siRNA or with scrambled siRNA control were treated with or without 200 μM I3C for the indicated durations. The relative amount of apoptosis was quantified by the ratio of sub-G1 DNA content as determined by flow cytometry. The number of apoptotic cells observed after I3C treatment was normalized to the number of apoptotic cells observed in DMSO-vehicle-control-treated cells. (B) 10AT-Her2 cells were transfected with either scrambled siRNA or nucleostemin-specific siRNA, and then treated with or without 200 μM I3C for 48 hours. Total cell extracts were immunoprecipitated with MDM2 antibodies, electrophoretically fractionated and Western blots probed with p53-specific antibodies. Negative control immunoprecipitations were carried out with non-immune antibodies (IgG) or samples that were not immunoprecipitated (No IP). (C) Western blots of total cell extracts (total protein) were analyzed for p53, serine-166 phosphorylated MDM2 and total MDM2. DMSO, dimethyl sulfoxide; I3C, indole-3-carbinol; IgG, immunoglobulin G; IP, immunoprecipitated; MDM2, murine double mutant 2; NS, nucleostemin; siRNA, small interfering RNA.