Specific knockdown of Hipk2 inhibits enucleation but not expression of erythroid-specific surface markers. Ter119-negative fetal liver cells infected with shRNA-containing retroviruses were cultured in Epo-containing media. Aliquots of cells from each sample were removed at 48 hours and stained with anti-Ter119 and anti-CD71 antibodies (to assay differentiation) or anti-Ter119 antibody and DAPI-Hoechst (to assay enucleation). (A) Forward scatter (FSC) versus side scatter (SSC) plots for control and Hipk2 knockdown cultures show no significant differences in the major cell populations. (B) Terminal erythroid differentiation as assayed by FACS; n = 4 (mean ± 2 SEM). (C) Extent of enucleation as assayed by FACS: enucleated cells (reticulocytes, circled), are defined by their lack of DNA but positive expression of Ter119, and are distinguishable from erythroblasts (solid arrow). Extruded nuclei (dashed arrow) have the lowest Ter119 signal but highest Hoechst signal because of their high DNA content and loss of membrane proteins. Quantification of enucleated cells is from 4 replicate experiments (mean ± 2 SEM). **P < .01.