PMC

Representative FACS analysis of MCF-7 cells, stable transfected with an expression vector coding for constitutive active Notch, or an empty vector (n = 2). Expression of constitutive active Notch increased the population of BCICs.

MCF-7, T47D, and MDA-MB-231 monolayer cells were serum-starved for 5 hours, trypsinized, and analyzed for EpoR-expression on the cell surface by flow cytometry. EpoR was expressed on CD44⁺/CD24^-/low cells, the putative stem cell population (gray) as well as on unselected cells (black).

Representative FACS analysis of Notch-1 activation in MCF-7 cells. Treatment with 1 IU/ml rhEpo for 2 or 4 hours (upper panel: gray line and black line; filled histogram, control) caused activation of Notch-1. This activation was inhibited by pretreatment with a GSI for 30 minutes at 2 and 4 hours (middle and lower panels: gray line, rhEpo; dashed line, rhEpo + GSI; filled histogram, control).

(A) Treatment of MCF-7 monolayer culture with 1 IU/ml rhEpo for 2 hours caused a 1.4 ± 0.16-fold (mean ± SEM, P = .041, n = 3, two-sided paired Student's t test) induction of Jagged-1 expression. (B) Treatment of MCF-7 monolayer culture with 1 IU/ml rhEpo for 2 hours caused a 1.5 ± 0.19-fold (mean ± SEM; P = .049, n = 3, two-sided paired Student's t test) activation of Notch-1 after 2 hours. Treatment with the PI3K inhibitor LY294002 but not the JAK2 inhibitor genistein prevented rhEpo-induced activation of Notch-1.

(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%).