Complete MammoCult medium was freshly prepared with 45 mL MammoCult basal medium (Stemcell Technologies, Canada), 5 mL growth supplement (Stemcell Technologies, Canada), 200 μL heparin adjusted to a final concentration of 4 μg/mL (Stemcell Technologies, Canada), 250 μL hydrocortisone adjusted to a final concentration of 0.48 μg/mL (Sigma, St. Louis, MO, United States), and 500 μL 100× penicillin and streptomycin (Invitrogen, Carlsbad, CA, United States). Complete MammoCult medium was filter-sterilized with a 0.22-μm syringe filter.
MCF-7 cells were collected and seeded in ultra-low attachment six-well plates (BD Biosciences, San Jose, CA, United States) with complete MammoCult medium at a density of 20,000 cells/well. Bakuchiol {4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl] phenol} purity 98% by HPLC was obtained from Enzo (Farmingdale, NY, United States). Cells were exposed to ethanol (vehicle control, 0.1% v/v, same amount in all the treatment conditions) or treated with 4 or 7 μg/mL Bakuchiol, then allowed to grow for a week to form mammospheres (p1). Mammospheres from each group were collected and trypsinized separately. Cells from different groups were seeded and treated with ethanol or Bakuchiol again for a week to form mammospheres (p2). Cell images were taken under an inverted microscope (model DMI3000 B, Leica Microsystems, Germany).
MCF-7 cells were collected and seeded in ultra-low attachment six-well plates (BD Biosciences, San Jose, CA, United States) with complete MammoCult medium at a density of 20,000 cells/well. Bakuchiol {4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl] phenol} purity 98% by HPLC was obtained from Enzo (Farmingdale, NY, United States). Cells were exposed to ethanol (vehicle control, 0.1% v/v, same amount in all the treatment conditions) or treated with 4 or 7 μg/mL Bakuchiol, then allowed to grow for a week to form mammospheres (p1). Mammospheres from each group were collected and trypsinized separately. Cells from different groups were seeded and treated with ethanol or Bakuchiol again for a week to form mammospheres (p2). Cell images were taken under an inverted microscope (model DMI3000 B, Leica Microsystems, Germany).