Figure 3

The inhibitory effect of RU017 and RU018 on MCF-7 mammosphere formation is dose-dependent. A) Photographs of mammospheres formed after six days incubation in anchorage-independent serum-free conditions. MCF-7 cells were seeded as a single cell suspension into media containing either (a) DMSO vehicle control or a range of concentrations of the halogenated monoterpene stereoisomers (i) RU017 or (ii) RU018 [(b)25, (c)50, (d)100 or (e)300 μM], (iii) fucoxanthin [(b)5, (c)10 or (d)15 μM] or (iv) paclitaxel [(b)50 or (c)100 nM]. The latter concentrations of compounds tested was informed by the cytotoxicity values observed under adherent conditions. Cells were photographed under a light microscope at 100× magnification. All images are representative of at least three randomly selected fields and were set to the same scale using ImageJ (NIH freeware). Scale bars representative of 0.1 mm. B) Quantification of the mammospheres generated in terms of sphere forming efficiency (SFE) after six days for the various treatments. SFE was calculated as the number of spheres (average diameter = 100 μm) formed in 96 wells plated with a single cell divided by the original number of single cells seeded and expressed as a percentage. C) Cell viability in treated mammosphere samples as assessed by WST-1 assay. The percentage viability after each of the treatments in C) was calculated relative to the DMSO-treated negative control (taken as 100%) after 8 days growth when an equal numbers of cells are seeded. Error bars indicate standard deviation where n = 3 (B) or 5 (C). Statistical significance of the differences in SFE and percentage survival relative to the DMSO control were calculated using a Students t-test (**p < 0.01, *p < 0.05).