Cholera toxin

All cell culture reagents were from ThermoFisher unless otherwise stated. N/TERT keratinocytes^[47 (link)
^] were provided by Prof. Michale Philpott's laboratory and were cultured in complete FAD medium, which comprised: 1 part F12 medium containing sodium pyruvate and GlutaMAX, 3 parts Dulbecco's Modified Eagle medium (DMEM) containing sodium pyruvate and GlutaMAX, 10% fetal bovine serum (FBS; Biosera Ltd), 1% penicillin/streptomycin (PS), 1.8 × 10⁻⁴ M adenine (Sigma Aldrich), 5 µg mL⁻¹ insulin (Sigma Aldrich), 0.5 µg mL⁻¹ hydrocortisone, 10 ng mL⁻¹ mouse epidermal growth factor (Peprotech Inc) and 1 × 10⁻¹⁰ M cholera toxin (Sigma Aldrich). N/TERT keratinocytes were passaged at 60–70% confluency and re‐seeded at a minimum density of 0.25 × 10⁵ cells mL⁻¹ and a maximum density of 1 × 10⁵ cells mL⁻¹.
Primary human dermal fibroblasts were obtained from residual tissue of elective abdominoplasty surgeries involving the removal of skin, with written patient consent under the East London and City Health Authority Research Ethics Committee approval (09/HO704/69) and were provided by Prof. Michael Philpott. Fibroblasts were cultured in DMEM containing sodium pyruvate and GlutaMAX and supplemented with 10% FBS and 1% PS. Fibroblasts were passaged once they reached 70–80% confluency and were re‐seeded at a minimum density of 2.5 × 10⁴ cells mL⁻¹, up to passage 10. To induce senescence, fibroblasts were treated with 0.3 mM H₂O₂ (Sigma Aldrich) in normal growth medium on two sequential days and then cultured for three additional days before seeding into microfluidic HSEs.
ECs (C2519A, LONZA) were cultured in Endothelial Growth Medium 2 (EGM2) containing SupplementMix (PromoCell) and 1% PS. The final concentration of the components within SupplementMix was: 2% FBS, 5 ng mL⁻¹ recombinant human epidermal growth factor, 10 ng mL⁻¹ recombinant human basic fibroblast growth factor, 20 ng mL⁻¹ recombinant human insulin‐like growth factor I (IGF‐I), 0.5 ng mL⁻¹ recombinant human vascular endothelial growth factor (VEGF), 1 µg mL⁻¹ ascorbic acid, 22.5 µg mL⁻¹ heparin, and 0.2 µg mL⁻¹ hydrocortisone. Cells were passaged at 90% confluency and were cultured to a maximum passage number of 5.
THP‐1 cells (TIB‐20, ATCC) were cultured in suspension in RPMI‐1640 culture medium supplemented with L‐glutamine, 10% FBS, and 1% PS. THP1 cells were cultured at a minimum density of 2 × 10⁵ cells mL⁻¹ and were passaged when reaching 8 × 10⁵ cells mL⁻¹. All cells were cultured at 37 °C, 5% CO₂.

Isolation of EpSCs from 3-day-old C57BL/6J mice was performed using standard protocols as described in previous studies (14 (link)). Briefly, the dorsal skin of the mice were incubated in 0.5% Dispase II solution (Roche; 04942078001, Switzerland) overnight at 4°C to separate the epidermis from the dermis. The epidermis was then dissociated into a single-cell suspension by gently pipetting up and down in 0.25% trypsin for 10 min. The trypsin was neutralized with calcium-free RPMI 1640 medium containing 10% FBS, and the cell suspension was then filtered through a 70-mm cell strainer to form a pellet. The cell pellet was then resuspended in a special EpSCs medium consisting of K-SFM (Gibco; 17005, USA) with 30 mg/mL bovine pituitary extract, 10 ng/mL mouse epidermal growth factor (BD, USA; 354001), 1×10^-10 M cholera toxin (Sigma; C9903), 0.05 mM calcium chloride, and 100 IU/L penicillin and streptomycin solution (Gibco; 15140122).
Cells were then counted and seeded onto dishes precoated with type IV collagen (Sigma; C5533) for 10 min at 37°C. Non-adherent cells were discarded by gentle washing with warm PBS, and the culture medium was changed every 2-3 days. Cells at passage 3 were used for subsequent experiments.

Primary human melanocyte strains Nohm1 (Bennett et al, 1985 (link)), 830c (Scott et al, 2002 (link)) and HM303CN (Minwalla et al, 2001 (link)) were grown as described (Bennett et al, 1985 (link)), except in the following melanocyte medium (Sviderskaya et al, 2003 (link)): RPMI 1640 medium, 10% foetal calf serum, 200 nM 12-O-tetradecanoyl phorbol 13-acetate (Sigma Chemical Co., Poole, UK), 200 pM cholera toxin (Sigma), 10 ng ml⁻¹ human stem cell factor (R&D Systems, Abingdon, UK) and 10 nM endothelin 1 (Sigma), gassed with 10% CO₂. Supplements shortly after retroviral infection also included 1 μM insulin, 40 pM fibroblast growth factor 2 (R&D Systems, Abingdon, UK) and 1 μg ml⁻¹α-tocopherol. Melanocytes for the BRAF studies only were obtained from Cascade Biologics and were grown in medium 254 with HMGS supplements (Cascade Biologics, Mansfield, UK). WM266.4 melanoma cells (ATCC), all producer cells and HeLa cells were grown in DMEM with 10% calf serum. Retroviral gene transfer into melanocytes was largely as described (Sviderskaya et al, 2003 (link)). All vectors, with or without inserts, were from Dr CJ Jones (Pathology, University of Wales College of Medicine, Cardiff, UK); the HPV16-E7 vector was originally from D Galloway (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). pBABEpuro or pBABEneo vectors were transfected into the Omega E ecotropic packaging line, and supernatant containing virus used to infect φCRIP amphotropic packaging cells (Danos and Mulligan, 1988 (link)). LXSN vectors were grown in PA317 amphotropic packaging cells (Halbert et al, 1991 (link)). φCRIP or PA317 medium containing infectious virus was harvested (using appropriate containment procedures) and frozen. Melanocytes were infected 1 day after plating them at 4–5 × 104 ml⁻¹. Supernatant was thawed, filtered (0.45 μm), supplemented with polybrene (3.5 μg ml⁻¹), and incubated overnight with melanocytes. Conditioned medium from the melanocytes before infection was then replaced. Normal culture was resumed after 2–3 days. For infection with a second vector, the procedure was repeated 2 weeks later. Inserts included hTERT cDNA (Geron Corp., Menlo Park, CA, USA) in pBABEpuro and HPV16-E7 in LXSN, as described before (Halbert et al, 1991 (link); Sviderskaya et al, 2003 (link)). The p16 antisense vector contained p16 exon 1α cloned in the antisense orientation between the BamH1 and EcoR1 sites of pBABEneo and expressed from the viral LTR. Normal human CDK4 cDNA was likewise expressed from the viral LTR of pBABEneo. Some melanocyte cultures following infection were plated on XB2 keratinocyte feeder cells (3 × 10⁴ ml⁻¹) (Sviderskaya et al, 2003 (link)).
Transient transfection was by Nucleofector™ technology, using the manufacturer's protocols (Amaxa GmbH, Cologne, Germany). 5 × 10⁵ melanocytes were transfected with 5 μg DNA of Myc-tagged pEFm vector containing either no insert, wild-type or human ^V600EBRAF sequences, and cultured in 1 ml normal medium in a well of a six-well plate for 5 days before assays.