Forced expression of wild-type Gfi-1B cDNA induces erythroid differentiation in UT7 and K562 cell lines. (A) Northern blot analysis of mRNA from UT7 and K562 cells transduced with full-length Gfi-1B or the empty vector MIGR. Gfi-1B cDNA was used as a ³²P-labeled probe. The expression level of the exogenous Gfi-1B is high (bicistronic mRNA of 5 Kb), while the endogenous expression level is low (2 Kb). (B) Measurement of cell proliferation by [³H]-thymidine incorporation. Two days after Gfi-1B transduction, the proportion of the cells in the S phase of the cell cycle was determined. Cells were incubated with [³H]-thymidine for 6 hours, and [³H]-thymidine incorporation was determined. Overexpression of Gfi-1B in K562 and UT7 cells inhibits cell proliferation in comparison with cells transduced with the empty vector MIGR. (C) Overexpression of Gfi-1B induces erythroid differentiation in both K562 and UT7 cells. GPA expression was evaluated by flow cytometry at day 4 after retroviral infection in UT7 cells (left). Hemoglobinization was determined by benzidine staining at day 6 in K562 cells (right). Full-length Gfi-1B expression induces expression of erythroid markers. Data are expressed as mean ± SD. (D) Globin expression analyzed by Northern blot using β, γ, and ϵ globin probes. Forced expression of full-length Gfi-1B induces expression of β and γ globins at a transcriptional level in UT7 cells in a same manner as EPO. In K562 cells, forced expression of Gfi-1B induces the transcription of the ϵ globin but not the β globin, in a same manner as AraC.