A.Epo phosphorylates ERK1/2 in a dose-dependent manner. Cells were starved for 1 hour and left unstimulated (C), incubated with vehicle (V), or increasing concentrations of Epo. Whole cell lysates were analyzed by immunoblotting using phospho-specific ERK1/2 antibody. The blots were stripped and re-probed with antibody against total ERK. Specific Epo-induced ERK phosphorylation(P-ERK) was further confirmed by additional controls (panels shown on right). Lane 1.untreated cells, Lane 2.vehicle, Lane 3.heat-denatured Epo(10units/ml), Lane 4.Epo pre-incubated with soluble EpoR antagonist prior to cell culture addition, Lanes 5-7.Epo treatment at indicated concentrations demonstrating increased ERK phosphorylation only in the presence of Epo. B.Time course of Epo-mediated phosphorylation of ERK1/2 in breast cancer cells. Cells were either left unstimulated (0) or stimulated with Epo (10units/ml) for the indicated time course. C.EPO-mediated induction of ERK kinase activity in breast cancer cells. Cells were either left untreated(−) or treated(+) with recombinant Epo (10units/ml) for 10 minutes at 37°C. Kinase assays were performed to measure ability of immunoprecipitated active ERK kinase to phosphorylate its substrate ELK-1. A representative immunoblot illustrating increased ELK-1 phosphorylation in response to Epo is shown(upper panel). Quantitative representation(mean±s.e.m) of fold-increase in ERK kinase activity(lower panel) in three independent experiments.*P< 0.05.

A.Growth curves of stably transfected MCF-7 cells cultured in the presence or absence of MEK inhibitor PD98059. Three independent single cell clones of empty vector- or EpoR-R129C expression plasmid-transfected cell lines were analyzed and the data was expressed as fold-increase in cell proliferation (n=9 replicates, *P<0.001 R129C compared to vector and to R129C-PD, #P<0.05 R129C compared to R129C-PD, **P<0.001 vector compared to vector-PD. B.Soft agar colony formation assays. Two independent single cell clones of empty vector or EpoR-R129C expressing cell lines were analyzed in the presence or absence of Epo (10units/ml) or MEK inhibitor for anchorage-independent growth with triplicate assays for each condition (n=11 replicates/group,**P<0.05,*P<0.001). C.Epo treatment or expression of EpoR-R129C promotes the migration of breast cancer cells. MCF-7 cells were placed in Transwell chambers in the presence or absence of JNK inhibitor SP600125 and Epo (1unit/ml). Three single cell clones in each group were analyzed for cell migration, the data was pooled and expressed as fold-increase over control(n=12/group,*P<0.001,**P<0.05).

A.MCF-7 cells stably transfected with empty vector or plasmid encoding EpoR-R129C were analyzed by immunoblotting for EpoR expression. Lane 1.negative control P769 cells without EpoR expression, Lane 2.anemic mouse spleen positive control, Lane 3.untransfected MCF-7 cells (UT), Lanes 4-6.Empty expression vector-transfected single cell clones, Lanes 7–9.EpoR-R129C transfected clones. The lower portion of the blot was analyzed for actin expression demonstrating protein integrity and comparable loading. Molecular weight marker(kDa) positions are illustrated. B.EpoR-R129C expressing cells exhibit increased proliferation compared to empty vector-transfected(Vector) or untransfected MCF-7 cells. The data of three independent clones was pooled and expressed as fold-increase in cell number to generate growth curves (mean±sem, n=8 replicates/group).**P<0.01 compared to vector-transfected or untransfected cells. C.Epo treatment (10units/ml) promotes the proliferation of vector-transfected but not EpoR-R129C expressing cells. Three single cell clones each of EpoR-R129C and vector-transfected cells were analyzed in triplicates, (n=9 replicates/group).*P<0.05 Epo-treated vector cells compared to no Epo treatment, #P<0.05 and **P<0.01 EpoR-R129C-expressing cells compared to vector-transfected cells without Epo treatment.

A.Increased basal phosphorylation of ERK (panels on left) and AKT (panels on right). Cells during exponential growth phase in RPMI and 10%FBS were removed from culture and whole cell lysates were analyzed for basal P-ERK and P-AKT by immunoblotting. Three independent single cell clones of empty vector and EpoR-R129C-transfected cells were analyzed. The blots were re-probed with total ERK and AKT antibodies to normalize phosphorylated protein amounts. Quantitative representation of P-ERK and P-AKT are illustrated below the blots.*P<0.05, n=3 clones/group. UT: untransfected MCF-7 cells. B-C.Epo-induced ERK1/2 (B), SAPK/JNK (C) and AKT (D) phosphorylation is increased in breast cancer cells expressing EpoR-R129C. Cells were starved for 1 hour and either left unstimulated(–) or incubated with increasing concentrations of Epo. Representative immunoblots are shown. Three independent single cell clones of empty vector or EpoR-R129C-transfected cell lines were analyzed. Epo-induced phoshorylation of the proteins was quantified by densitometry and normalized to total protein content. The data was pooled and expressed as fold-increase over control (mean±sem).#P<0.05,*P<0.01,**P<0.001 EpoR-R129C expressing cells compared to vector-transfected cells.