Mammary epithelium basal medium

A mammosphere assay was performed to determine cancer stem cell-like activity, as described in [46 ]. SKBR3 cells were seeded in ultra-low attachment plates (Corning) at a density of 500 cells/well in serum-free mammary epithelium basal medium (Lonza) supplemented with 1% penicillin/streptomycin (Lonza), B27 supplement minus vitamin A (50X, Gibco), 5 μg/mL insulin (Gibco), 1 μg/mL hydrocortisone (Sigma), 20 ng/mL EGF, and 20 ng/mL fibroblast growth factor (Sigma). Mammospheres were counted using an inverted microscope after 4 days of incubation in 37 °C, 5% CO₂. Mammosphere forming efficiency (MFE) was calculated as the number of mammospheres divided by the number of cells seeded per well and is expressed as a percentage.

HEK293T Phoenix (National Gene Vector Biorepository, Indianapolis, IN, USA) and NIH3T3 (ATCC; CRL-1658) cell lines were cultured in DMEM with 4.5 g/L glucose, L-glutamine, and sodium pyruvate (Corning, Glendale, AZ, USA) and supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, USA). BJ-5ta cell lines were cultured in DMEM supplemented with a 4:1 ratio of Medium 199 (Sigma-Aldrich, St. Louis, MO, USA) and 0.01 mg/mL hygromycin B (Invitrogen, Carlsbad, CA, USA) and 10% (v/v) FBS. MCF10A (ATCC; CRL-10317) cell lines were cultured in Mammary Epithelium Basal Medium (Lonza, Walkerville, MD, USA) supplemented with MEGM SingleQuots (Lonza) with the following modification: gentamicin sulfate-amphotericin was omitted from media, and cholera toxin (Sigma-Aldrich) was added at a final concentration of 100 ng/mL. All cells were grown at 37 °C with 5% CO₂ in a humidified incubator.

For mammosphere growth, cells were plated in ultra-low (Corning) tissue culture dishes at a density of 100–5000 cells/mL in a serum-free mammary epithelium basal medium (Lonza, Inc., Morristown, NJ) supplemented with B27 (Invitrogen, Carlbad, CA, USA), 1% antibiotic-antimycotic, 5 μg/mL insulin, 1 μg/mL hydrocortisone, 4 μg/mL gentamicin, 20 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA)), 20 ng/mL basic fibroblast growth factor (Sigma-Aldrich, St. Louis, MO, USA), and 1:25,000,000 β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Varying concentrations of celastrol and triptolide were added to the primary culture, whereas the second and third passages were grown in the absence of the drug. The number of mammospheres was counted under a Nikon eclipse microscope and the photos were acquired with NIS elements. For proliferation assay, 5000 cells were seeded on to 96-well plates and allowed to attach and grow overnight, following which they were treated with celastrol or triptolide. Analysis of cell proliferation was performed by hexosaminidase assay as previously described [24 (link)]. IC50 concentration was calculated using GraphPad Prism5. In all studies, the IC50 concentration was used unless mentioned otherwise.