Human recombinant epidermal growth factor

The WT VK2/E6E7 vaginal epithelial cell line was obtained from the ATCC (Manassas, VA, USA). This cell line (ATCC CRL-2616) was derived from normal human vaginal mucosal tissue and immortalized at passage 3 by transduction with the retroviral vector LXSN-16E6E7 in the presence of polybrene. The VK2 cells and the two TRIM26-modified VK2 variants (see below) were grown and maintained in keratinocyte serum-free medium (KSFM). KSFM was supplemented with an additional 0.1 ng/mL of human recombinant epidermal growth factor, 0.05 mg/mL of bovine pituitary extract, 0.4 mM CaCl₂, and 100 units/mL penicillin-streptomycin (Life Technologies, Carlsbad, CA, US), as described before [21 (link)]. KSFM, human recombinant epidermal growth factor, and bovine pituitary extract were obtained from Life Technologies. Human embryonic kidney (HEK) 293T cells containing the SV40 T-antigen (ATCC Manassas) were cultured in high glucose formulation of Dulbecco’s modified eagle’s medium (DMEM) with 4.5 g/L of glucose and supplemented with 10% fetal bovine serum (FBS). African green monkey kidney or Vero cells (ATCC) were cultured in minimum essential medium eagle-alpha modification (α-MEM) supplemented with 5% of FBS and 1% of l-glutamine, penicillin-streptomycin, and HEPES.

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)).

Human primary keratinocytes were purchased from Lonza. Keratinocytes were incubated at 37°C in humidified 5% CO₂ in serum‐free keratinocyte growth medium supplemented with human recombinant epidermal growth factor, bovine pituitary extract (5 μg/ml each), human insulin‐like growth factor I (1 μg/ml), and hydrocortisone (5 μg/ml; Gibco BRL).