Cholera enterotoxin

Dp was purchased from Mansite Bio-technology Co (Chengdu, China); DMEM/F12 medium and FBS were purchased from HyClone (Beijing, China); Trizol reagent, horse serum, gentamicin, insulin, Lipofectamine 2000, Opti-Mem were purchased from Invitrogen (Carlsbad, CA, USA); Epidermal growth factor (EFG) was purchased from PeproTech Inc (Rocky Hill, USA); PathScan Phospho-Akt ELISA assay kit and all antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[a]pyrene (B[a]P), cholera enterotoxin, hydrocortisol, dimethylsulfoxide (DMSO), phosphate buffered saline (PBS) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). All cell lines were purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China)

MCF7, MDA-MB231, HCT116, HeLa and HEK293T (from ATCC, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (BioWest, Nuaillé, France) as described^{35 (link)}. MCF10A cells were grown in DMEM/F12 medium (BioWest) with L-glutamine and HEPES, supplemented with 5% (v/v) horse serum, 100 μg/ml streptomycin and 100 U/ml penicillin from Gibco (Thermo Fisher Scientific, Waltham, MA, USA), and 20 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera enterotoxin and 10 μg/ml insulin from Sigma-Aldrich (St. Louis, MO, USA). hTERT RPE1 cells were grown in DMEM/F12 medium (BioWest) with L-glutamine and HEPES, supplemented with 5% (v/v) fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin from Gibco. NP40 extracts were prepared at 4 °C in 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1% Nonidet P-40 (NP40), 10% glycerol, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin and 10 μg/ml chymostatin for 20 min. Extracts were centrifuged at 20,000 g for 20 min and supernatants frozen in liquid nitrogen and stored at −80 °C. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA).

The study was carried out in an ER + human epithelial breast cancer cell line, MCF7, and in a human epithelial normal mammary cell line, MCF10A (American Type Culture Collection). MCF7 cells were cultured in DMEM/Ham’s F12 (1/1) medium (PAA Laboratories) supplemented with 10% (v/v) foetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 50 μg/ml streptomycin and 50 U/ml penicillin (Sigma-Aldrich). The human normal mammary epithelial cells MCF10A were grown in DMEM/Ham’s F12 (1/1) medium (PAA Laboratories) supplemented with 10% (v/v) horse serum (Gibco), 2 mM L-glutamine, 50 μg/ml streptomycin, 50 U/ml penicillin, 2.5 mg/ml insulin, 150 μg/ml cholera enterotoxin (Sigma-Aldrich), 2.5 mg/ml hydrocortisone and 50 μg/ml epidermal growth factor (Calbiochem). Both cell lines were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cells were routinely subcultured and they were always in exponential growth phase when used for experiments. Each experiment was independently performed at least in triplicate.

Tail skin was dissected and incubated in trypsin EDTA (Thermo Fisher, 0.25% in PBS) with the dermis side down overnight at 4 °C. The day after, the epidermis was peeled off and chopped with two scalpels for 1 min. Isolated cells were plated and cultured in keratinocyte medium (low-calcium Dulbecco’s modified Eagle medium (Thermo Fisher) supplemented with 10% FBS (Sigma-Aldrich), 100 U ml⁻¹ penicillin–streptomycin (Thermo Fisher), HCE cocktail consisting of hydrocortisone 0.5 μg ml⁻¹ (Sigma-Aldrich), insulin 5 μg ml⁻¹ (Thermo Fisher), cholera enterotoxin 10⁻¹⁰ M (Sigma-Aldrich) and EGF 10 ng ml⁻¹ (PeproTech) and cultured at 34 °C in a humidified atmosphere, with 8% CO₂.
For in vitro mini-bulk RNA-seq and ATAC–seq Sca-1⁺ tdTomato⁺ cells were sorted and cultured for 7 days over a feeder layer of mitomycin-treated NIH/3T3 cells. At day 7, cells were collected, and tdTomato⁺ were sorted to eliminate feeder cells and processed as indicated in mini-bulk RNA-seq and ATAC–seq paragraphs.

Primary HFKs were cultured in either M154 supplemented with 0.07 mM CaCl₂ or E-medium supplemented with 5 ng/ml mouse epidermal growth factor (EGF) (Becton Dickinson, Franklin Lakes, NJ; catalog no. 354010). E-medium is Dulbecco’s modified Eagle's medium (DMEM) supplemented with multiple factors essential for keratinocyte growth, including Ham’s F-12 (Life Tech), NaHCO₃ (Life Tech), penicillin-streptomycin (Pen-Strep; Life Tech), hydrocortisone (Sigma), cholera enterotoxin (Sigma), defined fetal bovine serum (FBS) (Sigma), adenine (Sigma), insulin (Sigma), transferrin, 3,3,5-triiodo-l-thyronine (T3; Sigma), and HEPES (Sigma) (35 (link)). All cells cultured in E-medium were cocultured on NIH 3T3-J2 fibroblasts arrested with 100 μl in 5 ml of mitomycin-c media (MEDAC) (0.4 mg/ml in PBS) for 1.5 h. CIN612 cells, which maintain the HPV 31 viral genome episomally, were cultured in E-medium plus EGF, with mitomycin-c-treated J2 feeders as well. J2 feeders were removed by incubating cells with Versene (PBS plus 0.05 mM EDTA) for 5 min and then rinsing the cells with PBS 2 to 3 times. 293T cells and PT67 Retropack J2 feeders were cultured in DMEM plus 1% penicillin-streptomycin (Life Tech) (5,000 U/ml) and 10% bovine serum (Life Tech). All cell lines were routinely tested for mycoplasma contamination.

Primary tumor cell lines were obtained from patients treated at the OHSU Department of Otolaryngology for HNSCC from 2014 to 2019 (Dermatology Molecular Profiling Resource Repository, IRB #10071). Cell culture methods have been previously described.^{24 (link),34 (link)} Primary tumor samples were collected from the operating room in DMEM/F12 Media, supplemented with 2x antibiotic/antimitotic (Invitrogen, Carlsbad, CA). Tissues were washed 3 times in fresh DMEM/F12 Media and minced before plating. Cells were cultured in DMEM/F12 Media (Gibco, 11320082), supplemented with 5% supplemented BCS (Hyclone), 1x antibiotic/antimitotic (Invitrogen, Carlsbad, CA), 1.8 × 10–4 M adenine (Sigma, St. Louis, MO), 0.4 μg/mL hydrocortisone (Sigma), 1 × 10–10 M cholera enterotoxin (Sigma), 2 × 10–9 M triiodothyronine (Sigma), 5 μg/mL insulin (Sigma), and 10 μg/mL epidermal growth factor (Invitrogen). Fibroblasts were removed from primary cultures by differential trypsinization using Trypsin-EDTA (0.25%), phenol red (Gibco, 25200056). Frozen cultured cell lines were used within five passages of the original tumor.