Cholera toxin

Normal human epidermal keratinocytes, strain NHEK-131 (GIBCO-Invitrogen, Paisley, UK), were derived from a pool of a minimum of three neonatal foreskins and obtained at 6.8 mean population doublings (MPDs). Keratinocytes were cultured at 37°C in a 10% CO₂/90% air with lethally irradiated 3T3 feeder cells in flavin-Adenine enriched medium (FAD^-). FAD^- consists of 3 parts DMEM 4.5 g/L glucose (Lonza, Slough, UK), 1 part Ham’s F12 (Lonza), 10% (v/v) Hyclone Fetalclone II serum (Fisher Scientific, Loughborough, UK), 20 mM HEPES buffer (Lonza), 100 U/ml penicillin, 100 U/ml streptomycin (Lonza) and 2 mM L-Glutamine (Lonza), supplemented with 1.8 × 10^-4 M Adenine (Sigma-Aldrich, Poole, Dorset, UK), 5 μg/ml insulin (Sigma-Aldrich), 5 μg/ml transferrin (Sigma-Aldrich), 0.4 μg/ml hydrocortisone (Sigma-Aldrich) and 8.4 ng/ml cholera toxin (Fisher Scientific, Loughborough, UK). Medium was replenished every third or fourth day with FAD⁺ complete medium, which consists of FAD^- supplemented with 10 ng/ml of epidermal growth factor (Sigma-Aldrich).

Human mammary tissue-derived cell lines were procured from American Tissue Culture Collection (ATCC, VA, USA). MCF10A cells (cat. # CRL-10317, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium supplemented with 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin, and 5% horse serum (Fisher Scientific). MCF7 (cat. # HTB-22, ATCC) and MDA-MB-231 (cat. # HTB-26, ATCC) cells were cultured in high-glucose DMEM supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific) and 10 μg/mL insulin (MCF7 only). All cells were cultured in 5% CO₂ at 37 °C. Replicate cell cultures were harvested at sub-confluence by scraping in 1x phosphate buffered saline. Cells were centrifuged at 7000 × g for 1 min, snap-frozen in liquid nitrogen and stored at −80 ^oC until use. Cell lysis was performed by resuspension and Dounce homogenization in buffer containing 10 mM Tris-HCl, 250 mM sucrose, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 0.1% (v/v) dodecyl-β-D-maltopyranoside (DDM) and 1× Complete Protease and Phosphatase Inhibitor Cocktail (Roche). The homogenates were treated with Turbonuclease (100 units/mL) (Accelagen) for 30 min at 4 ^oC, clarified by centrifugation at 18,000 × g for 20 min at 4 °C, quantified by Bradford assay (Bio-Rad) and adjusted to 6.0 mg protein/mL prior to fractionation.

MCF-10A parental cell lines were purchased from American Type Culture Collection (ATCC). MCF-10A cell line knock-ins were generated as previously described [15 (link)]. All cell lines were grown in 5% CO₂ at 37 °C with 1% Penicillin/Streptomycin in respective media conditions. Parental MCF-10A cell lines were cultured in DMEM/F12 (1:1) supplemented with 5% horse serum, 20 ng/ml epidermal growth factor (EGF), 10 µg/ml insulin (Roche), 0.5 µg/mL hydrocortisone (Sigma), and 100 ng/ml cholera toxin (Sigma). Knock-in cell lines were maintained in MCF-10A media in the absence of EGF. For all sequencing assays, cells were transferred to assay media 24 h prior to sample collection. Assay media contains phenol red-free DMEM/F12 (1:1) supplemented with 1% charcoal–dextran stripped FBS (Fisher), 0.2 ng/ml EGF, 10 µg/ml insulin, 0.5 µg/mL hydrocortisone, and 100 ng/ml cholera toxin.