Figure 4

Pre-treatment of MCF-7 cells with RU017/RU018 under adherent conditions is unable to prevent mammosphere formation. A) Treatment of MCF-7 cells was with a) DMSO vehicle control or either of the halogenated monoterpene stereoisomers b) RU017 or c) RU018 (100 μM), d) fucoxanthin (10 μM) or e) paclitaxel (100 nM). For pre-treatment, MCF-7 cells were cultured in regular anchorage-dependent culture conditions containing the compound of interest, before trypsinizing and transferring to anchorage-independent serum-free mammosphere conditions as a single cell suspension. The Day 0 and Day 4 samples refer to treatment of MCF-7 cells either upon seeding or after 4 days growth, respectively, in anchorage-independent serum-free culture conditions. Images were captured under a light microscope at 100× magnification. Each image is representative of at least 3 randomly selected fields. Images were set to the same scale using ImageJ (NIH freeware). Scale bars are equivalent to 0.1 mm. B) Quantification of mammospheres formed in terms of sphere forming efficiency (SFE) after six days for each treatment. 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) Assessment of cell viability in treated mammospheres 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. In both B) and C), error bars indicate the standard error of the mean 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).