The expression of GATA-1 in FDC2–wt-ER cells inhibits SCF mitogenic signaling and reduces c-kit transcript levels. (A) In FDC2–wt-ER and derived FDC2-ER/G1 cells, mitogenic responsiveness to Epo and SCF was assayed based on cytokine-stimulated reduction of MTS. For FDC2-ER/G1 cells, polyclonal (left panel) as well as derived clonal lines (right panel) were analyzed. (B) In FDC2–wt-ER and FDC2-ER/G1 cells, levels of c-kit transcripts were analyzed by Northern blotting. Total RNA (20 mg per lane) was used, and equivalence in loading was confirmed using a cDNA probe for 7S RNA.33 Cell lines analyzed include parental control FDC2–wt-ER, FDC2-EE483, FDC2-EE372 cells,26 and FDC2-ER/G1 cells (polyclonal, “poly” clonal lines, −7 and −12).

(C) Ectopic expression of GATA-1 in FDC2–wt-ER cells is associated with the transcription of endogenous GATA-1, EKLF and β^maj genes. Based on the above-observed inhibition of SCF signaling in FDC2–wt-ER cells expressing GATA-1 (ie, FDC2-ER/G1 cells) possible effects on lineage commitment were investigated. Total RNA was isolated from FDC2, FDC2–wt-ER, and FDC2-ER/G1 cells and levels of ectopically expressed Epo receptor mRNA, ectopically expressed GATA-1 mRNA, and endogenous GATA-1, EKLF, and β^maj-globin transcripts were assayed by Northern blotting. GATA-1 mRNA expressed from a pXM vector is larger than, and resolves from, endogenous GATA-1 transcripts. In polyclonal FDC2-ER/G1 cells (FDC2-ER/G1.Poly) expression of endogenous genes for GATA-1, EKLF, and β^maj-globin was activated. In contrast, no detectable levels of transcripts for GATA-1, EKLF, or β^maj-globin were observed in control FDC2 or FDC2–wt-ER cells. As a positive control, RNA from murine erythroleukemic SKT6 cells also was analyzed at two levels of loading (1X, 1/3X; left most lanes).