Down-regulation of SHIP-1 in HB60-5 cells blocks differentiation and accelerates proliferation. (A) RNAi-mediated down-regulation of SHIP-1 in HB60-5 double-sorted population (dbl sort) and clone 8D cell populations results in increased Fli-1 expression and increased phosphorylation of AKT and MAPK compared with nonspecific negative control shRNA expressing cells (control). (B) Fli-1 protein expression is reduced in the presence of the PI 3-K inhibitor, LY294002, but not the MAPK inhibitor, PD169316. (C) Northern blot analysis of RNAi-induced SHIP-1 knockdown cells displays reduced Epo-induced differentiation as determined through hybridization with an α-globin probe. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Double-sorted SHIP-1 shRNA expressing HB60-5 cells (SHIP-1 shRNA-11), grown in the presence of Epo, display accelerated proliferation, compared with negative control shRNA-expressing cells. Cells were maintained in 1 U/mL Epo. This experiment was performed in triplicate.