Nuclear relocation of NPM by leptomycin B in primary NPMc⁺ AML cells. (A-B) Bone marrow from an NPMc⁺ patient with AML bearing NPM mutation A. (A) Leukemic cells show cytoplasmic (in addition to nuclear) expression of NPM; endothelial cells of a vessel (lumen indicated with an asterisk) show nucleus-restricted NPM (Olympus 100×/1.3 NA UPlanFl oil objective). (B) Leukemic cells, with the exception of a mitotic figure (arrow), show the expected nucleus-restricted expression of C23/nucleolin (Olympus 100×/1.3 NA UPlanFl oil objective). (C-E) Purified leukemic cells from the patient were immunostained with a monoclonal anti-NPM antibody (clone 376) and observed at the confocal laser microscope (100×/1.4 NA PlanApochromat oil objective). Images were reconstructed in 3 dimensions, and an electronic cut of the nuclear surface was performed to better analyze the localization of NPM. The antibody 376, which cannot distinguish between the wild-type and NPM mutant A proteins (both contained in the leukemic cells), labels both nucleoli and cytoplasm in the absence of LMB. (F-H) Incubation with LMB results in nucleoplasmic relocation of cytoplasmic NPM.