Expression of epithelial-mesenchymal transition (EMT)-associated markers in the total cell population and the cycling hypoxia-selected subpopulation. (a) Three-color fluorescence-activated cell sorting (FACS) analysis was performed to detect CD44CD24 expression profile on the E-cadherin-negative cell population. Unstained, isotype control, and E-cadherin (E-cad)-stained cells are shown in the histogram. ESA staining of MDA-MB 231 (total cell population) and MDA-MB 231 F3 (cycling hypoxia-selected subpopulation) cells is shown. M1 marker gates E-cadherin-positive cells, and M2 marker gates the E-cadherin-negative cells. Bottom panel: CD44 and CD24 expression of E-cadherin-negative cells in each cell line. The percentages of the CD44+/CD24- and CD44+/CD24+ cells are indicated in the Quad plot. (b) Quantitative comparison of E-cad-/CD44+/CD24- cell population and E-cad-/CD44+/CD24+ cell population in cycling hypoxia-selected subpopulations (MDA-MB 231 F3 and BCM2 F3) and their parental breast cancer cell lines. At least three replications were performed to derive the average percentage of each cell population in each cell line and standard deviations among the replicates. (c) Expression of EMT-associated proteins in the parental cells (P), hypoxia-exposed adherent cells (A3), and the cycling hypoxia-selected subpopulations (F3). Lysates from these two cell populations were fractionated on the basis of the subcellular localization using the ProteoExtract Kit, and 40 μg of proteins was loaded for Western blotting. Beta-actin is used as the loading control for the cytoplasmic fraction, and histone H3 is used as the loading control for the nuclear fraction. Western blot images (n = 3) are quantified by National Institutes of Health ImageJ software and presented as fold increase in A3 and F3 cells compared with the parental cell lines. (d) mRNA regulation of EMT transcription factors in the parental, A3, and F3 cells. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses were performed using specific primers to measure the mRNA expression of human Snail, Slug, and Twist genes in these three cell populations. At least three replications (n = 3) were performed to derive the average percentage of each cell population in each cell line and standard deviations among the replicates. (e) Expression of EMT-suppressing microRNAs (miRNAs) in the parental, A3, and F3 cells. qRT-PCR analyses were performed using specific primers to measure the expression of miR200c and miR205. At least three replications (n = 3) were performed to derive the average percentage of each cell population in each cell line and standard deviations among the replicates. Asterisks indicate statistical significance by two-tail t test (P < 0.05) for all quantitative results. ESA, epithelial-specific antigen.