The migration of nuclear proteins such as histones out of the nucleus during normal erythroid differentiation is inhibited by Xpo7 knockdown. (A-B) Immunofluorescence of cultured erythroblasts after 48 hours of culture containing shRNA against either (A) control or (B) Xpo7. Red staining is against histone 2A using an Alexa-594 secondary antibody and blue staining is against DNA (DAPI). Note that there is less red staining overlying the nuclei in the control shRNA-infected cells and increased accumulation in the cytoplasm, but in contrast, after Xpo7 knockdown, histone staining remains colocalized with the nucleus. In all panels, scale bar = 10 μm. (C) Histogram of amount (pixels per cell) of cytoplasmic, or noncolocalized with DAPI, red staining in cultured control (red) or Xpo7-KD (blue) cells at 48 hours. Mean noncolocalized red staining (pixels per cell) for 35 cells is depicted as a dotted line (control, 8.49; shXpo7, 4.00). There is significantly more staining outside of the nucleus in control cells compared with Xpo7-KD cells (Student t test, P < .001). (D) Immunofluorescence of cultured erythroblasts after 48 hours of culture containing shRNA against either control or Xpo7 and a plasmid containing a histone H2A-mTurquoise fusion protein. Red staining shows nuclear DNA using ToPro dye, and turquoise depicts localization of the fusion protein. There is less turquoise staining over the nucleus in control shRNA-infected cells (and more in the cytoplasm), whereas in Xpo7-KD cells, red and turquoise staining colocalize.