Myeloid progenitor–expressing RUNX1 mutants fail to differentiate into granulocytes. (A) Expression of exogenous RUNX1 proteins was detected by WB using RUNX1 polyclonal Ab in 32D cells. (B) Differentiation induction by G-CSF in all 4 indicated stable cell lines. Wright staining of 32D cell lines at day 0 or day 8 of G-CSF treatment. (C) The percentage of 32D cells in 3 different stages of myeloid differentiation is indicated (day 8). (D) The expression profile of MPO and LF mRNA was examined by semi-quantitative RT-PCR in the 4 indicated 32D cell lines with G-CSF treatment for 0, 3, 6, 8, and 10 days. (E) Expression pattern of Gr-1 reveals maturational arrest of 32D cells with RUNX1 mutants. The expression profiles of Gr-1 of 32D cell lines at day 0 or day 8 of G-CSF treatment are shown. (F) Growth properties of 32D stable cell lines. Left, all 4 cell lines were grown in the indicated conditions, and cells were counted each day. The 32D cell lines expressing the mutated RUNX1 protein continue to proliferate when cultured in the presence of G-CSF, whereas the cell lines transfected with vector and WT RUNX1 construct stopped proliferation 5 days after G-CSF treatment. Error bars represent SD. Right, growth curve of the 14 days after withdrawal of G-CSF. Cells expressing either of the 2 mutants kept growing after replacing G-CSF with IL-3, whereas growth of cells with control vector or WT decreased rapidly. Error bars represent SD. (G) Quantification of the colonies for 32D stable cell lines in methylcellulose. 32D cell lines at day 0 or day 8 of G-CSF treatment were plated in methylcellulose medium containing IL-3. Colonies were counted after incubation at 37°C for 14 days.