(A) Activation of cell signaling by rHuEPO in human breast cancer cell lines. The indicated cells were left untreated or were treated with 10 U/ml rHuEPO for 30 or 120 minutes in low-serum medium. Cell lysates were prepared, and equal amounts of cell lysates were subjected to Western blot analysis with antibodies directed against total and activation-specific phosphorylated Akt, Erk, and STAT5. The ratios represent quantitative analysis of densitometric values of specific band intensities normalized to the value of the corresponding untreated controls, which was arbitrarily set at 1. The level of β-actin served as a protein-loading control.
(B) Dependence of rHuEPO-induced activation of cell signaling on EpoR expression. MCF7 cells were transfected for 72 hours with a control vector or one of two shRNA constructs targeting different regions of EpoR. The cells were stimulated with 10 U/ml rHuEPO for 30 minutes and immediately lysed for Western blot analysis with the indicated antibodies.
(C) Effect of rHuEPO on cell signaling in additional breast cancer cell lines. The indicated cell lines were treated and analyzed as described in (A).