p19^INK4dknockdown causes abnormal nuclear morphology of erythroblasts and ectopic GATA1 expression can reverse the abnormal phenotype. (A) Representative images of Lucif shRNA or p19^INK4d shRNA-infected erythroblasts (day 15 cells) and quantitative analysis of cells with abnormal nuclei. (B) Flow cytometry analysis of sorted annexin V⁻ erythroblasts (left). Representative images of sorted annexin V⁻ erythroblasts (right). (C) Representative images of sorted erythroblasts at distinct development stages after Lucif shRNA or p19^INK4d shRNA infection (left). Quantitative analysis of abnormal nuclear morphology of sorted erythroblasts at the polychromatic and orthochromatic stages from 3 independent experiments (right). (D) Representative images of erythroblasts infected with Lucif shRNA, p19^INK4d shRNA, and p19^INK4d shRNA combined with GATA1 overexpression (day 15 cells) (left). Quantitative analysis of nuclear morphology data from 3 independent experiments (right). (E) Representative images of western blotting showing GATA1 expression in erythroblasts transfected with GATA1 siRNA or scramble siRNA (day 15 cells) (left). Quantitative analysis of expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative images of erythroblasts (day 15 cells) transfected with GATA1 siRNA or scramble siRNA (left). Quantitative analysis of nuclear morphology data from 3 independent experiments (right). The percentage of abnormally nucleated cells = counts of abnormally nucleated cells in 2000 cells/2000. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. **P < .01, ***P < .001.