Different biologic effect induced by ECD and PTD mutants in Rat2 and EML cells. (A) Colony formation by Rat2 cells expressing different KIT mutants in soft-agar medium. Cells were plated at a concentration of 3 × 104 per dish under the indicated growth condition. Colonies were photographed (top panel) under inverted microscopy (original magnification ×4) and counted (bottom panel) at day 10. Data are mean ± SD of duplicates. (B) Epo-induced hemoglobinization of EML cells bearing different KIT. EML parental cells, EML-WT cells (with 5 U/mL Epo and mSCF), and KIT mutants expressing EML cells (with 5 U/mL Epo) were grown in erythroid differentiation medium for 12 days. Hemoglobin-positive cells were scored after benzidine staining. Data are percentage of benzidine-positive cells in a field. (C) Epo-induced BFU-E formation by KIT variants expressing EML cells. Different EML cells mixed with 1 mL methylcellulose were seeded in 6-well plates in the presence of 10 U/mL Epo and/or mSCF. Colonies were counted after 7 days. Data are percentage of BFU-E compared with total colonies. Results represent the mean ± SD of duplicates. (A-C) Results are representative of 3 independent experiments.
