Fungizone antimycotic

NIH/3T3 cells (a gift from Margaret Goodell) were cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum, 100 U ml⁻¹ of penicillin, 100 μg ml⁻¹ of streptomycin and 250 ng ml⁻¹ of Fungizone antimycotic (Invitrogen). Media was replaced every 2 days and cells were subcultured twice per week using 0.25% (w/v) Trypsin–0.53 mM EDTA solution. In all, 5 × 10⁵ cells were nucleofected with 300 ng total DNA in P3 Primary Cell Solution (Lonza) using the 4D Nucleofector program EN-158. Cells were collected after 48 h and genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). PCR amplification was performed using Phusion polymerase (NEB, cat# M0530L). Primer sequences are given in Supplementary Fig. 1.

Cervical loops were dissected under a Leica M80 stereomicroscope. Explants were incubated in 1% type I Collagenase (Sigma‐Aldrich) in HBSS with 1% penicillin–streptomycin and 1% Fungizone^® Antimycotic (all from Thermo Fisher Scientific) for 60 min and dissociated by pipetting 20 times every 15 min. Collagenase was deactivated with equal amount of DMEM containing 10% fetal bovine serum (Sigma‐Aldrich) and 1% penicillin–streptomycin. The cell suspension was then centrifuged at 1,000 × g for 5 min, and the resulting pellet was re‐suspended in DMEM/F12 (Thermo Fisher Scientific) containing B27 supplements (Minus vitamin A version, Thermo Fisher Scientific), recombinant mouse EGF (20 ng/ml final concentration; carrier‐free form, R&D Systems), recombinant mouse basic FGF (25 ng/ml final concentration; carrier‐free form, R&D Systems), and 1% PS. From the second passage, cells started to express typical keratinocyte‐like morphology. The exclusion of contamination of fibroblasts was confirmed by real‐time RT–PCR on Cytokeratin 14 and Vimentin (Appendix Fig S2E). All experiments were performed with cells between passages 4 and 10. For SHH stimulation experiments, cells were treated with indicated concentration of SHH protein (carrier‐free form, R&D Systems) for 24 h before further analysis.

Primary bovine dermal fibroblast cells were isolated from visibly healthy bovine foot skin tissue as previously described (Evans et al., 2014 (link)). Isolated cells were seeded into 25 cm³ tissue culture flasks at 2 × 10⁴ viable cells per ml in growth media. Williams’ medium E (WME; Sigma-Aldrich, Poole, UK) was supplemented with 100 µg/ml neomycin (Sigma), 50 µg/ml gentamycin (Sigma), 20% (v/v) foetal bovine serum (Gibco™ by Thermo Fisher Scientific, Loughborough, UK), 2 mM L-glutamine (Gibco™), 2.5 µg/ml Fungizone^® Antimycotic (Gibco™), 10 ng/ml human recombinant epidermal growth factor (Gibco™). Cultures were maintained within a humidified incubator (37°C, 5% CO₂) to passage eight with subculture (Evans et al., 2014 (link)).

Histopathological analyses confirmed the presence of tumor cells in the samples used for culture. Five PeCa samples were dissociated and plated as previously described [10 (link)]. Briefly, minced tumor fragments were seeded in 25 cm² culture flasks containing 3:1 KSFM (keratinocyte serum-free medium)–DMEM/F12 (Dulbecco’s modified Eagle medium/nutrient mixture F-12) (GIBCO, Carlsbad, CA, USA) added with 2.5% fetal bovine serum (FBS) (HYCLONE, Waltham, MA, USA), 30 µg/mL of bovine pituitary extract (BPE) (GIBCO, Carlsbad, CA, USA), 0.2 ng/mL of epithelial growth factor (EGF) (GIBCO, Carlsbad, CA, USA), antibiotics (100 IU/mL penicillin G and 100 mg/mL streptomycin) (Sigma-Aldrich, St. Louis, MO, USA), and 25 µg/mL of Fungizone^® Antimycotic (GIBCO, Carlsbad, CA, USA). After confluence, cells were treated with 0.05% trypsin/0.02% EDTA (Sigma-Aldrich, St. Louis, MO, USA) and replicated for at least 10 passages (P10).

RAW 264.7 murine macrophage cells were obtained from American Type Culture Collection (ATCC), and were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum and 1% Antibiotic-Antimycotic (10,000 units/ml of penicillin, 10,000 μg/ml of streptomycin, and 25 μg/ml of Fungizone® Antimycotic, GIBCO). Cells were seeded on a 6-well plate in complete growth medium one day before infection experiments at a density of 1x10⁶ viable cells/well. Murine Norovirus genotype 1 (MNV-1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA), and was propagated in RAW 264.7 cells. Virus titers were determined by plaque assay on RAW 264.7 cells [33 (link)]. S. enterica serovar Heidelberg isolate 163 [30 (link)] was grown in LB broth. A viable cell count, as a function of an optical density standard curve, was generated and used to determine cell inoculum for infecting RAW 264.7 cells. Post-infection bacterial titers were determined by plating serial dilutions on Tryptic Soy Broth with 1.5% agar plates.