NFI-A regulates erythroid (E) versus granulocytic (G) lineage differentiation in bilineage E + G culture (mean ± SEM values from 3 independent experiments). (A) Ectopic expression of NFI-A promotes E and inhibits G differentiation/maturation. (i) Growth curve, (ii) morphology analysis of the cellular composition at sequential culture times, and (iii) differentiation/maturation of the E population of vector- and NFI-A–infected HPCs. (B) NFI-A knockdown blocks E and promotes G differentiation/maturation. (i) Growth curve, (ii) morphology analysis at sequential culture times, and (iii) differentiation/maturation of the G population in culture of vector- and siNFI-A–infected HPCs. (C) Lineage-specific marker expression at early stage (day 9) of culture. (i) Representative flow cytometry analysis of vector-, siNFI-A–, and NFI-A–infected cells using the erythroid GPA and the myeloid CD14/CD15 markers, (ii) β-globin and G-CSFR mRNA detected by real-time PCR, and (iii) percentage of cells expressing G-CSFR detected by flow cytometry (day 7). (D) Macroscopic and morphologic changes of vector-, siNFI-A–, and NFI-A–infected cells at day 9 of E + G culture. (Top) Macroscopic view of cellular pellets centrifuged from vector culture (mixed population, erythroid red cells in the center and peripheral myeloid-white cells in the surrounding ring), siNFI-A culture (only myeloid cells), and NFI-A culture (predominance of red cells). (Bottom) Representative morphology fields (original magnification, ×400). See “Morphologic analysis” for more image information. (E) E + G clonogenic activity of HPCs transduced with vector, siNFI-A, or NFI-A viruses. Histograms represent the relative GFP⁺ BFU-E and CFU-G colony distribution. GFP⁺ colony numbers were: vector BFU-E 34.3 ± 7, CFU-G 14.3 ± 5; siNFI-A BFU-E 4.6 ± 1.5, CFU-G 31 ± 9; NFI-A BFU-E 29.3 ± 6, CFU-G 3.5 ± 1 (mean ± SEM values from 3 paired experiments).