Expression of TEL-Syk led to increased STAT5 phosphorylation and promoted colony formation independent of JAK2 activity.

<p>(A) Lysates of sorted GFP⁺ and GFP^- fetal liver cells were separated by SDS-PAGE and immunoblotted with indicated antibodies. (B) Representative histograms of retrovirally transduced fetal liver cells cultured for 3 days in cytokines (IL-3, IL-6, SCF), starved for 6 hours, then restimulated with these cytokines. Cells were fixed, permeabilized and levels of phospho-STAT5 in the GFP⁺ cells was determined by flow cytometry. (C) Normalized MFI of phospho-STAT5 in retrovirally transduced fetal liver cultured for 3 days in cytokines, followed by a 6 hour starvation and 30 minute restimulation. Phospho-STAT5 MFIs were normalized to vector expressing cells. (D) Colony formation of sorted GFP+ fetal liver cells in methylcellulose in the presence of a JAK2 inhibitor. Colony numbers were normalized to a DMSO-treated (ie no inhibitor) control for each cell type and represented as a percent of maximal growth after 7 days in culture in 10 ng/mL GM-CSF. (E) Histograms of sorted GFP⁺ fetal liver cells starved for 6 hours and restimulated for 30 minutes with or without 50 ng/mL GM-CSF in the presence of the JAK inhibitor 1 at the indicated concentrations, stained for phospho-STAT5 and assessed by flow cytometry. Experiments were conducted in triplicate. Statistical significance was determined by one-way ANOVA. ***P< 0.001. Data is representative of 3 independent experiments.