Representative p53 localization patterns in primary AML cells from case 14, which were treated with 10 μM Nutlin-3a for 3 hours. Cells were stained for p53 (green) and mitochondrial marker protein cytochrome c oxidase IV (red) and visualized by confocal microscopy. Nuclei were counterstained with DAPI (blue). Localization of p53 to mitochondria is indicated by the yellow-orange color in the merged images. (A) Untreated cells showed low levels of diffusely distributed p53. (B) After treatment, individual cells showed either cytoplasmic, cytoplasmic and nuclear, or nuclear accumulation of p53. In this case, a majority (75%) of cells showed cytoplasmic accumulation of p53, and the inhibitory effect of cycloheximide on loss of Δψ_m compared with Nutlin-3a alone was 26%. A preferential translocation of cytoplasmic p53 to mitochondria suggests that cytoplasmic p53 mediates apoptosis mainly at the level of mitochondria.