Emem medium

The human adipose-derived mesenchymal stem/stromal cells (ADMSC) and TNBC-derived cell line MDA-MB-231 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). ADMSC were grown in Mesenchymal Stem Cell Basal Medium (ATCC, PCS-500-030) and supplemented with Mesenchymal Stem Cell Growth Kit—Low Serum (ATCC, PCS-500-040). They were further reported to have the capacity to undergo adipogenesis [27 (link)]. MDA-MB-231 were grown in EMEM Medium (Wisent, 320-036-CL) supplemented with 10% of fetal bovine serum. All cells were cultured at 37 °C under a humidified 95–5% (v/v) mixture of air and CO₂. The TNBC cells secretome was generated upon a 48 h serum deprivation of a ~70% confluent MDA-MB-231 culture. Next, the conditioned media (CM) was harvested and centrifuged at 1500× g for 20 min to eliminate cell debris. CM was aliquoted and kept at −20 °C. To evaluate the induction of the CAA phenotype, ADMSC were cultured with the TNBC cells secretome in the presence or absence of 10 μM EGCG for 24 h. Then, cells were collected for total RNA extraction, protein isolation, or cell migration studies.

Caco-2/15 cells were cultured at 37 °C and 5% of CO₂ in EMEM medium (Wisent Inc., Canada) containing 10% fetal bovine serum (FBS; Wisent Inc.), 1% penicillin/streptomycin and 1% non-essential amino acids (GIBCO, USA) as described previously^{64 (link),65 (link)}. Cells were maintained in T-75 cm² flasks (Corning Inc., USA) until they reached 80–90% of confluence and were trypsinized. For individual experiments, enterocytes were seeded at the density of 1 × 10⁶ cells/well on polycarbonate 24.5-mm transwell filters with pores of a diameter of 0.4 µm (Corning Inc., USA). Differentiation occurred when enterocytes were cultured for 15 days post-100% confluence.

CFBE-ΔF508 and CFBE-wt cell lines [CFBE41o- parental cells (Kunzelmann et al., 1993 (link)) stably transduced, respectively, with F508del-Cftr and wt-Cftr Bebok et al., 2005 (link)] were grown in EMEM medium (Wisent Inc., St-Bruno, QC, CA) supplemented with 10% FBS (Life technologies, Burlington, QC, CA), 2 mM L-glutamine (Life technologies) and 100U/ml of penicillin-streptomycin (Life technologies) on 35 mm petri dishes (Corning Inc., Corning, NY, USA), Lab-Tek 8 chamber slides (Thermo-Fisher Scientific Inc., Waltham, MA, USA) and permeant filters (4.67 cm²; Corning Inc.) coated with a LHC basal medium (Life technologies) solution containing 1 mg/ml BSA (Life technologies), 0.05 mg/ml bovine collagen I (Life technologies) and 1 mg/ml human fibronectin (VWR, Mont-Royal, QC, CA). Cells were cultured for 5 days on Lab-Tek chamber slides for immunofluorescence assays and 8 days on petri dishes before protein extraction, while cells on permeant filters were cultured for 3 weeks before electrophysiological measurements (Trinh et al., 2012 (link), 2015 (link); Bilodeau et al., 2016 (link)).