Characterization of UT-7/GM subclones. (A) Growth profiles of UT-7/GM subclones to several cytokines. UT-7/GM cells were subcloned by picking up a single colony formed in the semisolid culture containing methylcellulose medium. Each colony was expanded in liquid suspension culture in the presence of GM-CSF (1 ng/mL). Proliferative responses of UT-7/GM subclones (E-6 and F-4) to cytokines were analyzed by the MTT assay (see Materials and Methods). Cells were plated at a density of 10⁴/well in IMDM supplemented with 5% FCS and cultured with medium alone (lane 1), EPO (10 U/mL; lane 2), TPO (100 ng/mL; lane 3), or GM-CSF (10 ng/mL; lane 4). MTT reduction was measured after 3 days of culture. The values represent the mean ± SD from triplicate cultures. (B) The effect of EPO or TPO on the erythroid or megakaryocytic differentiation of E-6 (a and c) and F-4 (b and d). After complete removal of GM-CSF from the conditioned media, EPO (10 U/mL; a and b) or TPO (100 ng/mL; c and d) was added to the liquid culture. One week later, the cells were obtained and prepared for analysis of cell-surface markers (GPA & GPIIb/IIIa) by immunofluorescence. Thin line, treatment with GM-CSF; thick line, treatment with EPO or TPO.