Bakuchiol

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).

RPMI-1640 medium (11 mM glucose and L-glutamine) and fetal bovine serum were purchased from Gibco (Paisley, UK). Penicillin/streptomycin antibiotic mixture and Dulbecco's phosphate-buffered saline (DPBS) were purchased from WELGENE (Daegu, Korea). 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Duchefa (Haarlem, Netherlands). Primary antibodies against poly(ADP-ribose) polymerase (PARP) (9542), caspase-3 (9662), bcl-2 (2876), bax (2772), p62 (5224S), and beclin-1 (3495) were purchased from Cell Signaling Technology (Beverly, MA, USA), and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, sc-2004; anti-mouse, sc-2005) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Psoralen, isoPsoralen, palmitate, rapamycin, and bovine serum albumin were obtained from Sigma-Aldrich (St Louis, MO). Bakuchiol was purchased from Enzo Life Sciences Inc. (Farmingdale, NY).

Dulbecco modified eagle medium (DMEM), Ham’s F-12 (F-12) medium, and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Bakuchiol was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). Antibodies against poly (ADP-ribose) polymerase (PARP), B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter (Bad), and phospho-Bad were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against beta-actin and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Streptozotocin (STZ), losartan potassium, psoralen, and isopsoralen were obtained from Sigma-Aldrich (St. Louis, MO, USA).