0.4 μm polyester membrane

HEK293T cells (ATCC, CRL-3216^TM) were cultured in DMEM supplemented with 10% fetal bovine serum, 0.3 mg/ml L-glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin at 37°C in 5% CO₂. The cells were transfected by the calcium phosphate method. MTECs were isolated and cultured as described before [24 (link)]. Briefly, MTECs were isolated from 8-week old C57BL/6J mice and were freshly seeded onto transwells with 0.4-μm polyester membrane (Corning, NY 14831). 10 μM DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester) (Sigma), an inhibitor of the Notch signaling pathway, was added to increase the percentage of multiciliated cells [25 (link)].

Co-culture was performed using a Transwell system. Breast cancer cells were seeded (3.5 × 10⁴ SUM159PT cells or 10⁵ SK-BR-3 cells) in the inserts (0.4 μm polyester membrane; Corning Life Sciences, Lowell, MA) and cultivated alone or with mature adipocytes in the bottom chamber. In some cases, adipocytes were substituted for 0.02% Intralipid (Sigma–Aldrich) for 3 days.

Caco-2 cells stably transfected with different vectors (details see Additional file 4: Supplementary Methods) were seeded at 10,000 cells/cm² on transwell filters with 0.4-μm polyester membrane (Corning) and allowed to grow for 20 days to develop a monolayer. The cells were cultured with complete medium containing 2.5 ng/mL tumor necrosis factor-alpha (TNFα; PeproTech) and 10 ng/mL interferon-gamma (IFN-γ; PeproTech) [52 (link)]. The TEER was measured on day 21. The resistance value (ohms, Ω) was detected by Millicell ERS-2 Voltohmmeter (Millipore, Darmstadt, Germany), and the TEER was calculated using the following formula: $$TEER\left(\mathrm{\Omega },\bullet ,{\mathit{cm}}^2\right)=\left(\mathrm{measured}\mathrm{ohms},-,\mathrm{blank}\mathrm{insert}\mathrm{ohms}\right)(\mathrm{\Omega })\times \mathrm{filter}\mathrm{area}({\mathrm{cm}}^2)$$
Paracellular permeability was assessed by FD4 (Sigma-Aldrich) flux through the caco-2 cell monolayer, as previously described [53 (link)]. Opti-MEM medium (Gibco) supplemented with FD4 (2 mg/mL) was added to the upper compartment on day 21, as described in the TEER procedure. Next, Opti-MEM medium was added to the lower compartment and incubated for 4 h at 37 °C; 200 μL of media was collected from the lower compartment to detect FD4 fluorescence intensity using a spectrophotometer (SpectraMax M5, Molecular Devices, CA, USA).