Mouse epidermal growth factor

mOct4⁻ BM-MAPCs were obtained from the Stem Cell Institute at KU Leuven. Cells were cultured as described previously [29 (link)]. In short, dishes coated with fibronectin were used when splitting the cells every 48 hours. Medium contained 60 % low-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Sigma-Aldrich, St. Louis, MO, USA), 40 % MCDB-201 (Sigma-Aldrich), 1 × selenium-insulin-transferrin-ethanolamine (ITS; Sigma-Aldrich), 0.2 mg/ml LA-BSA and 0.8 mg/mL powdered bovine serum albumin (BSA; Sigma-Aldrich), 10⁻⁴ M ascorbic acid 3-phosphate (Sigma-Aldrich), 100 units of penicillin, 1000 units of streptomycin (Gibco®, Invitrogen, Carlsbad, CA, USA), 2 % fetal bovine serum (Gibco®, Invitrogen), 10 ng/mL human platelet-derived growth factor (R&D systems, Minneapolis, MN, USA), 10 ng/mL mouse epidermal growth factor (Sigma-Aldrich), 1000 units/ml mouse leukemia inhibitory factor (Esgro®, Millipore, Billerica, MA, USA) and 1 × chemically defined lipid concentrate (Gibco®, Invitrogen). Finally, β-mercaptoethanol (1 ×; Gibco®, Invitrogen) was added freshly to the media before sterilization with a 22-mm filter (Millipore).
The GL261 cell line, extensively used in mouse models of glioblastoma, was obtained from Dr S. Van Gool, KU Leuven. GL261 cells were cultured as described earlier [30 (link)].

The Vero (ATCC) and HaCaT (obtained from Dr. Meenhard Herlyn, Wistar Institute, Philadelphia, PA) cell lines were maintained using standard procedures in DMEM supplemented with 10% fetal bovine serum and Gentamicin. Primary mouse keratinocytes were isolated from 24 to 72 h old pups^{20 (link)}. The pup tail tips were boiled in 10 mM NaOH, 0.1 mM EDTA, neutralized with 1 M Tris, pH 8.0 and used for PCR typing of sex with primers SMCX-1, 5′-CCGCTGCCAAATTCTTTGG-3′ and SMC4-1 5′-TGAAGCTTTTGGCTTTGAG-3′. These primers generate 1 product from female cells and 2 products from male cells. Skins were floated in 0.25% Trypsin (Invitrogen) overnight at 4 °C. The epidermis and dermis were separated and keratinocytes from the epidermis dissociated by mechanical shearing. Keratinocytes were maintained in a 1:1 mix of Keratinocyte-SFM with calcium and Keratinocyte-SFM without calcium (Invitrogen) supplemented with 10 ng mL⁻¹ mouse epidermal growth factor (Sigma-Aldrich), 200 μg mL⁻¹ bovine pituitary extract (Sigma-Aldrich) and Gentamicin. Experiments were performed with independent duplicate samples per timepoint and treatment. These duplicate samples were analyzed separately.

The PCa cell lines LNCaP (derived from metastatic site—left supraclavicular lymph node) and DU145 (derived from metastatic site—brain) was obtained from ATCC and grown in RPMI-1640 medium (Gibco, Life Technologies, Carlsbad, USA), supplemented with 10% fetal bovine serum (FBS; gibco, 10270-106). The PCa cell line MDA-PCa2b (derived from metastatic site—bone) was obtained from ATCC and cultured in F-12K medium (Gibco, Life Technologies, Carlsbad, USA), supplemented with 20% none heat-inactivated fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, USA), 25 ng/mL cholera toxin (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL mouse Epidermal Growth Factor (Sigma), 0.005 mM phosphoethanolamine (Sigma), 100 pg/mL hydrocortisone (Sigma), 45 nM sodium selenite (Sigma), and 0.005 mg/mL human recombinant insulin (Sigma). The radioresistant properties of LNCaP RR cells were validated prior to experiments. The cell lines were cultured in a humid atmosphere at 37 °C and 5% CO₂. Every batch tested negative for mycoplasma contamination. The cell lines were genotyped using microsatellite polymorphism analyses.

Trigeminal ganglia (TG) were removed from thy1-YFP mice and cultured as previously described [20 (link), 23 (link)]. In brief, ophthalmic branches of the trigeminal nerves were harvested and cleaned from contaminating tissue, then subjected to enzymatic digestion with papain and collagenase/dispase (Worthington Biochemicals, Lakewood, NJ) and separated in Percoll gradients by centrifugation. Cell cultures were maintained in 96-well plates coated with 20 μg/mL laminin (Sigma, St. Louis, MO) and supported with media (Neurobasal; Invitrogen, Carlsbad, CA) supplemented with 2% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (ABAM; Gibco, Grand Island, NY). Mouse corneal epithelial sheets were isolated using dispase treatment, as described by Kawakita et al.[24 (link)]. Epithelial sheets were rendered into single cells by 0.25% trypsin-EDTA (Gibco). Dissociated cells were seeded in six-well plates with corneal epithelium growth medium (Epilife; Cascade Biologics, Portland OR) with 20 μM calcium, 1% human corneal growth supplement (Cascade Biologics), 0.01 μg/mL mouse epidermal growth factor (Sigma), 0.1 nM cholera toxin A subunit from Vibrio cholerae (Sigma), and 1% ABAM (Gibco) until they were 90% confluent.