Effects of dominant negative constructs on colony formation induced by Flt3-ITD.
(A) Equal amounts of an empty control vector and a dominant negative STAT5 (Y694F) were electroporated into 32D cells containing ITD1. One day after electroporation, cells were plated at a concentration of 1 × 105 cells per dish in the presence of 0.6 mg/mL G418 and no cytokine. Numbers of colonies were counted at day 8. Data shown are representative of 3 independent experiments. (B) Inducible expression of dn-Ras. ITD1/dnRas were polyclonal cell lines, which were made by transfection of Flt3-ITD1 into the 32D/dnRas clone. Cells were treated with 0.5 mmol/L IPTG for 24 hours, and total cell lysates were subjected to Western blot with anti-Ras Ab. These cells were subjected to a colony assay as described in Figure 4, under the presence or absence of IPTG.
