Figure 5
FOXM1 increases expression of cancer stem cell markers and colony formation and invasiveness. (A) FOXM1 levels impact the percentage of the CD44+/CD24−/low population in MCF-7 cells. CSC, Cancer stem cell-like cells; KD, Knockdown; OE, Overexpression; TamR, Tamoxifen-resistant cells. (B) Increased levels of the marker CD44 by fluorescence-activated cell sorting (FACS) analysis. FACS analyses were done in three separate experiments, and a representative profile from one FACS run is shown. Counts indicate number of events (that is, cells detected). Ctrl, Control. (C) Western blot obtained after FOXM1 overexpression. (D) Expression of CD44 mRNA during exposure to Tamoxifen and the development of TamR cells. (E) FACS profile of MCF-7 and TamR cells with ABCG2-gated population. FSC-H, Forward scatter height. (F) ABCG2 expression levels during the time course of development of TamR cells. (G) Proliferation and 4-hydroxytamoxifen (OH-Tam) response with and without knockdown of ABCG2 in TamR cells. OD, Optical density. (H) Schematic representation of the separation of ABCG2+ and ABCG2- populations in TamR cells. FITC, Fluorescein isothiocyanate. (I) ABCG2+ and ABCG2- cell populations from TamR were sorted by FACS and monitored for proliferation in response to several concentrations of OH-Tam with and without FOXM1 knockdown. (J) FACS analysis of estrogen receptor α (ERα) levels in ABCG2+ and ABCG2- cell populations in TamR cells. (K) Percentage of Hoechst dye excluding side population (SP) cells in the overall TamR cell population or in ABCG2+ and ABCG2- cells after 12 days of culture. (L) Image of soft agar assay and three-dimensional Matrigel spheroid formation in TamR, ABCG2+ and ABCG2- cells. (M) Number of colonies formed in the soft agar assay and (N) Diameter of the colonies from TamR cells or separated ABCG2+ and ABCG2- cells. *P < 0.05; **P < 0.01.