Effects of dominant-negative forms of Stat1α and Stat3 on the inhibition by GM-CSF of EPO-induced erythroid differentiation. (A) UT-7/GM cells were transfected with a plasmid DNA mixture (1 μg of reporter genes containing 4 copies of APRE in front of the minimaljunB promoter linked to the luciferase gene, 2 μg of either expression vector pCAGGS-Neo, with no insert or with an insert of Stat cDNA encoding either HA-Stat3F or HA-Stat1F, and 1 μg of pSV-β-galactosidase. After transfection, the cells were cultured without growth factors and then stimulated with GM-CSF (10 ng/mL) for 6 hours. Luciferase values were normalized for β-galactosidase activity and expressed relative to the normalized luciferase activity in the extracts from unstimulated cells transfected with the reporter plasmids and a control expression plasmid. The data are the mean ± SD of more than four independent experiments. (B) MTT reduction assay. Cells were plated at a density of 10⁴/well in IMDM supplemented with 5% FCS and cultured with GM-CSF (10 ng/mL). MTT reduction was measured after 3 days of culture. The data are the mean ± SD of triplicate cultures. (C) Dianidisine staining. Cells were cultured in the presence of GM-CSF (10 ng/mL) or a combination of EPO (10 U/mL) and GM-CSF (10 ng/mL). Seven days later, cells were harvested for dianisidine staining. The data are the mean ± SD from three independent clones.