The colony formation and proliferation of the parent cell lines and the cycling hypoxia-selected subpopulations. (a) Images of colony formation assays from 500 cells of MDA-MB 231, MDA-MB 231 A3, MDA-MB F3, BCM2, BCM2 A3, and BCM2 F3 on days 1, 3, 5, and 9. Representative images from three separate experiments are shown. (b) Colony-forming efficiency (CFE) of parental cells, hypoxia-exposed adherent cells (MDA-MB 231 A3 and BCM2 A3), and the cycling hypoxia-selected subpopulations (MDA-MB 231 F3 and BCM2 F3). CFE was determined when tumor spheres were larger than 60 μm in diameter on day 9 after plating. CFE was calculated by dividing the number of tumor spheres formed by the original number of single cells seeded and is expressed as a percentage. Three independent experiments were performed to derive the average CFE and standard deviation per cell line. Asterisk indicates statistical significance by two-tail t test (n = 3, P < 0.05). (c) Proliferation curves of the parental cells, hypoxia-exposed cells (A3), and the cycling hypoxia-selected subpopulations grown as adherent cultures (top two plots) or as spherical cultures (the third plot). Total viable cells from each well at days 3 and 5 were counted by the ViaCount assay using the Guava Flow Cytometer. Average cell numbers and standard deviations at each time point were calculated from three independent experiments.