Figure 5

The halogenated monoterpenes RU017 and RU018 have a dose-dependent inhibitory effect on second and third generation mammosphere formation. Untreated mammospheres of first and second generation were dissociated and reseeded into anchorage-independent conditions in serum-free culture media containing the compounds of interest in order to assess second (A) and third generation (B) sphere forming efficiency (SFE), respectively. In both cases, mammosphere-derived MCF-7 cells were seeded as a single cell suspension into media containing either the DMSO vehicle control or either 100 or 300 μM of the halogenated monoterpene stereoisomers RU017 or RU018. SFE was calculated as the number of spheres (average diameter = 100 μm) formed after six days in 96 wells plated with a single cell divided by the original number of single cells seeded and expressed as a percentage. Error bars indicate the standard error of the mean where n = 5. Statistical significance of the differences in SFE relative to the DMSO control were calculated using a Students t-test (**p < 0.01, *p < 0.05).