Specific knockdown of Hipk2 reduces hemoglobin accumulation. GFP⁺ cells were sorted from fetal liver erythroid cultures grown in Epo-containing media 36 hours after shRNA retroviral infection. (A) Slides made from cytospins of infected cells were fixed and stained with benzidine-Giemsa (original magnification 40×). Positively stained cytoplasm reflects the presence of hemoglobin: the more differentiated orthochromatophilic erythroblasts (thick arrow) show more hemoglobin staining than the remaining early erythroblasts (thin arrow). (B) Quantification of positively stained, hemoglobinized cells, expressed as the percentage of stained cells in 5 unique 40× fields for each sample; n = 5 (mean ± SEM). **P < .01. (C) Equal numbers (1 × 10⁶) of GFP⁺ cells were lysed with 200 μL Drabkin reagent and spectrophotometric readings made at 540 nm; n = 5 (mean ± SEM). **P < .01; ***P < .001.