Gata2 expression during terminal differentiation of βLCR-G2::G1KO:Y and HRD-G2::G1KO:Y fetal liver cells. βLCR-G2 line A was used. (A) Size distribution of TER119⁺ wild-type, βLCR-G2::G1KO:Y, and HRD-G2::G1KO:Y cells after 2 days in hanging drop cultures. The percentage of small, enucleated cells is indicated. Significant difference: *, P < 0.05; **, P < 0.01; compared with WT (unpaired t test). (B) RQ-PCR analysis of gene expression. RNA was isolated from βLCR-G2::G1KO:Y fetal liver cells, and gene expression was determined with real-time quantitative RT-PCR as described previously.²⁸  Bar graphs depict higher (white area) or lower (gray area) expression levels relative to WT fetal liver cells; error bars indicate ± SD (n ≥ 3 for each gene measured). Significant difference: *, P < 0.05; **, P < 0.01 compared with WT (unpaired t test). (C) RT-PCR detecting the distal and proximal exon 1 of endogenous Gata2 mRNA; RNA from dendritic cells is a positive control for detection of distal exon 1. (D) Top panel: Western blot of βLCR-G2-and HRD-G2-derived Gata2 in Gata1-null cells during erythroid differentiation; expression of Gata1 and Gata2 in HRD-G2 cells wild type for endogenous Gata1 is shown for comparison. Bottom panel: detection of Npm1 serving as a loading control.