(A) FACS analysis detecting the size of the CD44+/CD24-/low cell population in monolayers and supernatant of MCF-7 cells. Treatment with rhEpo (3 x 1 IU/ml) increased the size of this population 4.8-fold from 2 ± 0.08% to 9.5 ± 1.8% (P < .01, Student's t test). Data from four independent experiments are shown. (B) Representative FACS analysis of MCF-7 monolayer cultures treated with rhEpo (3 x 1 IU/ml) for 3 consecutive days. About 0.9% of the cell in the adherent cell population was CD44+/CD24-/low (left panel). Treatment with rhEpo increased the frequency of CD44+/CD24-/low cells in the supernatant from 2% (middle panel) to 14.9% (right panel). (C and D) Primary sphere formation assay of MCF-7 (C) or MDA-MB-231 (D) cells treated with rhEpo (3 x 1 IU/ml) for 3 consecutive days. RhEpo increased the rate of primary spheres formation (MCF-7: 1 ± 0.4% for untreated cells, 2.9 ± 0.6% for Epo-treated cells; MDA-MB-231: 1.2 ± 0.6 for untreated cells, 4.5 ± 1.1% for Epo-treated cells, P < .01, two-sided Student's t test; means ± SEM). Preincubation (30 minutes) with GSI (5 µM) treatment prevented the rhEpo-induced increase in primary sphere formation (MCF-7: GSI-treated cells, 1 ± 0.4%; Epo + GSI-treated cells, 1.5 ± 0.4%; MDA-MB-231: GSI-treated cells, 1.5 ± 0.7%; Epo + GSI - treated cells, 1.1 ± 0.8%).