Dmf12

The CRC cell lines Colo205, HCT116, and HT29 were purchased from the American Type Culture Collection, USA; Caco2 and JEG3 cells were purchased from The European Collection of Cell Cultures (ECACC). Prior to experiments, all cell lines were authenticated by short tandem repeat profiling and were used between passages 10 and 35. Furthermore, all experiments were performed during the exponential growth phase of the cell line. HCT116 and HT29 were routinely cultured in McCoy’s 5a modified medium (Gibco, Life Technologies, USA) with 10% v/v heat inactivated FBS (Sigma-Aldrich, UK). Colo205 cells were culture in RPMI with 10% FBS; Caco2 cells were cultured in MEM with 10% FBS. JEG3 cells were cultured in DM-F12 (Gibco, USA) with 10% FBS. All culture mediums were supplemented with 2 mM L-glutamine (Sigma-Aldrich, UK) and 1% PenStrep (Gibco, USA). JEG3 cells were used as control as they exhibit high STS activity.
For experimental conditions, cells were initially starved of estrogens for 72 h by placing them in their appropriate phenol-red free medium plus 10% charcoal stripped FBS (sFBS) (Sigma-Aldrich, UK). After starvation, HCT116 and HT29 cells were treated with E₂ (100 nM), G1 (100 nM), G15 (1 μM), Tamoxifen (10 nM) or fulvestrant (1 μM) in stripped medium for 24 h prior to measuring STS activity.

Adipose tissue was rinsed thrice with PBS and then minced and digested with 0.25% collagenase type I (Gibco, Carlsbad, CA, USA) at 37 °C for 1 h. The mixture was then added to the complete medium (CM; DM/F12, 10% fetal bovine serum, 2% penicillin-streptomycin) (Gibco) followed by filtration through 70-nm and 40-nm cell sieves, respectively. The final mixture was centrifuged at 1200×g for 5 min to collect the preadipocytes. The preadipocytes were then seeded into a culture flask with complete medium and incubated at 37 °C in a humidified incubator with 5% CO₂. Culture medium was changed every 2 days and the cells were frozen for future studies. After the cells reached about 70% confluence, an adipogenic cocktail (0.5 mM 3-isobutyl-1-methylxanthine, 10% FBS, 1 μM dexamethasone, and 1.7 μM insulin) was added into the growth medium to induce differentiation. The cells were further incubated for 72 h, after which the medium was replaced with maintenance medium (growth medium supplemented with 1.7 mM insulin per 50 mL) and incubated for an additional 72 h. Thereafter, the cells were cultured in a growth medium until maturation at 10 days.