FACS of breast cancer cell lines and characterization of their differentiation status. (a) Expression of EpCAM and CD49f in MCF-7 (luminal), SUM149PT (basal-like) and SUM159PT (claudin-low) cell lines. The gates shown in each cell line (gray squares) represent the different sorted subpopulations that were further evaluated. (b) Differentiation scores of the different cell sorted subpopulations. Means and SD are shown for each subpopulation. Only significant P values (P < 0.05) are shown. (c) Gene expression analyses of the two FACS-sorted subpopulations within SUM149PT. A paired two-class SAM (FDR < 5%) was performed between both subpopulations in three independent experiments. (d) In vitro differentiation of CD49f+/EpCAM-/low SUM149PT cells. The two SUM149PT sorted cell subpopulations were grown in vitro under the same conditions as before FACS. After 7-11 days in culture, expression of CD49 and EpCAM was reanalyzed in both subpopulations using FACS. Blue, MCF-7-sorted cell fractions; red, SUM149PT CD49f+/highEpCAM+-sorted subpopulation; orange, SUM149PT CD49f+/EpCAM-/low-sorted subpopulation; yellow, SUM159PT-sorted cell fractions. Similar results were obtained with and without supplemental FBS in the SUM149PT cell line.