Fig. 1.

a Effect of 200 μM fatty acid on T47D invasion. T47D cells, obtained from the American Type Culture Collection, and found to exhibit the exquisite progestin-responsiveness characteristic of these cells, were grown in plastic flasks in 5 % CO₂ in air in minimal essential medium, powdered (autoclavable) plus non-essential amino acids, 2 mM l-glutamine, 10 % fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 6 ng/ml insulin (Sigma-Aldrich) until about 90 % confluence. The medium was then changed to fresh and made 200 μM in EPA, AA, or 0.1 % in ethanol (vehicle). Cells were incubated for 72 h, and the medium was made 10 μM in ara-C (arabinofuranosyl cytidine) for the last hour, to stop DNA replication. EPA, AA, and ara-C were purchased from Sigma-Aldrich. The cells of each flask were then harvested and single cell suspensions at 10⁶ cells/ml (300 μl) in the same medium as above (containing ara-C), except without serum and phenol red and with or without 10 nM R5020 (dissolved in ethanol) were placed in the upper insert of a modified Boyden chamber (Cell Biolabs, catalog # CBA-110) to measure invasion through a layer of extracellular matrix and a membrane with 8-μm pores, to a lower chamber containing 500 μl of twice charcoal-stripped serum-containing medium without phenol red. Final ethanol concentration was 0.2 % in all samples. As stated above, the cells were grown to around 90 % confluency in complete growth medium (containing 10 % fetal bovine serum and phenol red), and then incubated with the tested fatty acids in fresh complete growth medium for 72 h. They were then made into single cell suspensions and incubated in the presence of the fatty acids with or without progestin for a further 48 h in the same medium as above, except serum-free and without phenol red, while invading through a layer of extracellular matrix and a porous membrane into 10 % charcoal-stripped serum-containing medium without phenol red. After 48 h, invading cells were stained and counted, according to the manufacturer’s instructions. Results are the average plus SEM of six independent experiments, and were analyzed by ANOVA followed by the Fisher’s LSD multiple comparison procedure. CC no fatty acid or R5020, RC R5020 alone, CE EPA alone, RE R5020 plus EPA, CA arachidonic acid alone, RA R5020 plus arachidonic acid. All samples are different from control (CC) at p < 0.05 except for CA. All other samples are statistically the same except RC, CE, and CA (p < 0.05). *Different from control. **Different from RC and control. ***Different from RC. b Effect of 75 μM fatty acid on T47D invasion. Protocol was the same as in (a), except that fatty acid concentration was 75 μM. Results are the average plus SEM of nine independent experiments for CC and RC, and three experiments for the remaining samples. Results were analyzed by ANOVA followed by the Fisher’s LSD multiple comparison procedure. Abbreviations are the same as in (a). All samples are different from control at p < 0.05. *Different from all others. No other differences are significant. c Effect of 40 μM fatty acid on T47D invasion. Protocol was the same as in (a), except that fatty acid concentration was 40 μM. Results are the average plus SEM of nine experiments for CC and RC, and three experiments for the remaining samples. Abbreviations are the same as in (a). Results were analyzed by ANOVA followed by Fisher’s LSD multiple comparison procedure. *Different from all others at p < 0.05