rHuEpo treatment of tumor cell lines cells did not lead to increased phosphorylation of signaling proteins. Tumor cell lines were starved of serum and rHuEpo overnight. Cells were stimulated for 5 minutes with vehicle (rHuEpo formulation buffer), a rHuEpo titration from 0.02 to 300 U/mL, or an EGF/HGF/IGF-1 growth factor cocktail (EGF 100 ng/mL, HGF 500 ng/mL, and IGF-1 500 ng/mL). UT-7/Epo cells were the positive control and COLO677 the negative control for rHuEpo treatment. Fixed and permeabilized cells were stained with fluorochrome-conjugated antibodies to the phosphorylated forms of the proteins, run on a FACS instrument, and analyzed as fold-change compared with vehicle treatment alone. Experiments were repeated 3 times with similar results. (A) Growth factor cocktail (EGF/HGF/IGF-1) 5-minute stimulation of 6 tumor cell lines analyzed for p-AKT. Note the stimulation of p-AKT in response to the growth factor cocktail in the tumor cell lines. (B) UT-7/Epo, NCI-H661, and COLO677 treated with increasing concentrations of rHuEpo and analyzed for p-AKT. (C) UT-7/Epo, NCI-H661, and COLO677 treated with increasing concentrations of rHuEpo and analyzed for p-STAT5. Note the lack of response in NCI-H661 cells with p-AKT and p-STAT5. (D) Effect of 5-minute stimulation of NCI-H661with 300 U/mL rHuEpo on phosphorylation of 4 signal transduction proteins. Similar results were seen after 30 minutes (data not shown).