FIGURE 5.

Nuclear mortalin causes strong inactivation of p53. A, schematic diagram of mortalin constructs. sp, signal peptide. B, overexpression of mortalin and its p53-binding mutants inhibited etoposide-induced nuclear translocation of p53. Double immunostaining with anti-p53 (green) and anti-mortalin (red) antibodies showed nuclear translocation of p53 in control, mot-A mutant-transfected etoposide-treated cells. Mot-F-, mot-N-, and mot-B-transduced cells showed cytoplasmic retention of p53, as seen by cytoplasmic staining that is colocalized with mortalin and, hence, seen as yellow. C, quantitation of cells with etoposide-induced nuclear translocation of p53. D, coimmunoprecipitation (IP) of mot-F and mot-N with p53. Immunoprecipitated mortalin-myc by anti-myc antibody (Myc-IP) was examined for the presence of p53. Control immunoprecipitation was performed with isotype-matched IgG. Quantitation of p53 precipitated with mortalin-myc is shown. E, a p53-dependent reporter assay showed a stronger inactivation of p53 activity in mot-N-transduced cells. Luc, luciferase. F, coimmunostaining of mortalin and Aurora A revealed a stronger intensity of coimmunostaining (yellow signal) of the two proteins in MCF7/mot-N than MCF/mot-F cells. G, coimmunostaining of mortalin-myc and γ-tubulin showing the presence of aneuploid cells in mot-F and mot-N cells. The cell aneuploidy and anomalies score was higher in mot-N cells.