Mammocult serum free medium

MCF10A or MCF10DCIS cells were grown to 50–60% confluence and cells were detached with StemPro Accutase (Life Technologies). Cells were then plated at 10,000 cells/mL in 6-well ultra-low attachment plates and maintained in Mammocult serum-free medium supplemented with hydrocortisone and heparin (Stem Cell Technologies). Cells were treated with 1α25(OH)₂D₃ (100 nM) or BXL0124 (10 nM). For secondary and tertiary mammosphere culture, primary mammospheres were collected and enzymatically dissociated using StemPro Accutase (Life Technologies). Then, cells were re-plated at a density of 5,000 cells/mL for subsequent passages. Photos of mammospheres were taken, and the number of mammospheres was counted to determine the mammosphere forming efficiency (MFE). The MFE was calculated by dividing the number of mammospheres (≥100 μm) formed by the number of single cells seeded. Roundness of spheres was obtained by analysis of photos with ImageJ software (US National Institutes of Health, Bethesda, MD). The formula used to calculate roundness is 4 × ([area]/π[major axis]²). A value of 1.0 represents an object that is perfectly round. A value of 0.0 represents an object that is formless. Experiments were repeated in triplicates.

SUM159 cells were grown to 50-60% confluence and cells were detached with StemPro Accutase (Life Technologies, CA). Cells were then plated at 2,000 cells/mL in 6-well ultra-low attachment plates and maintained in Mammocult serum-free medium supplemented with hydrocortisone and heparin (Stem Cell Technologies, Vancouver, Canada). Cells were treated with 1α,25(OH)₂D₃ or BXL0124 for five days for each passage. For secondary and tertiary mammosphere culture, primary mammospheres were collected and enzymatically dissociated using StemPro Accutase (Life Technologies, CA). Then, cells were re-plated at a density of 2,000 cells/mL for subsequent passages. Images of mammospheres were taken, and the number of mammospheres was counted to determine the mammosphere forming efficiency (MFE). The MFE was calculated by dividing the number of mammospheres (≥100 μm) formed by the number of single cells seeded. Experiments were repeated three times.