Transferrin

SILAC labelling was performed as previously described [15] (link), [16] (link), briefly: NHF were cultured and passaged in SILAC-DMEM with 4.5 g/L glucose, sodium pyruvate and 3.7 g/L NaHCO₃ (PAN Biotech), supplemented with 10% dialyzed FBS (Gibco), 1% penicillin/streptomycin, 82 mg/L l-proline, 84 mg/L l-arginine HCl (Arg0) and 146 mg/L l-lysine HCl (Lys0) for light labelling (Sigma-Aldrich). For medium-heavy labelled cells, Arg0 and Lys0 were replaced by l-arginine-¹³C₆¹⁴N₄ and l-lysine-²H₄ (Arg6, Lys4). For heavy labelling, l-arginine-¹³C₆¹⁵N₄ and l-lysine-¹³C₆¹⁵N₂ (Arg10, Lys8) were used.
SCC13 cells were cultured in SILAC-Keratinocyte Growth Medium 2 (KGM2) without calcium, l-arginine, and l-lysine (PromoCell), supplemented with SupplementMix containing 0.004 mL/mL bovine pituitary extract, 0.125 ng/mL epidermal growth factor, 5 μg/mL insulin, 0.33 μg/mL hydrocortisone, 0.39 μg/mL epinephrine, 10 μg/mL transferrin and 0.06 μM CaCl₂ (PromoCell), with 1% penicillin/streptomycin. l-Lysine HCl concentrations were the same as mentioned above. For l-arginine HCl, 210 mg/L were used [17] (link).

We cultivated primary human skin fibroblast lines (HF40 and HFF-1) (n = 2 each) that were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum and, when confluent, medium was supplemented with or without recombinant human (rh)-IL-17 (R&D System, Minneapolis, MN) of 200 ng ml⁻¹ (same IL-17 source and concentration used in prior experiments with human keratinocytes) [10] (link), [12] (link). After 24-hour incubation, fibroblasts were harvested for further analyses.
We also cultivated NHEKs obtained from PromoCell, in the Keratinocyte Growth Medium 2 supplemented with 0.004 ml/ml BPE, 0.125 ng/ml EGF, 5 ug/ml Insulin, 0.33 ug/ml Hydrocortisone, 0.39 ug/ml Epinephrine, 10 ug/ml Transferrin, and 0.06 mM Ca++ (all items purchased from PromoCell GmbH, Heidelberg, Germany). The experiment was performed in triplicate.
Once 70–80% confluent, the medium was changed with full media containing 0.06 mM Ca++, 1.2 mM Ca++, or 1.2 mM Ca++ plus 2.0% FBS, for 24 and 48 hours before harvesting for other analyses.

The human keratinocyte cell line hTert/KER-CT (ATCC^®, CRL4048) was purchased from LGC Standards GmbH (Wesel, Germany) and cultured as described [44 (link)]. The cells were maintained at 37 °C and 5% CO₂ in basal Keratinocyte Growth Medium 2 (KGM2, PromoCell, Heidelberg, Germany) supplemented with 4 µL/mL bovine pituitary extract, 0.125 ng/mL epidermal growth factor (recombinant human), 5 µg/mL insulin (recombinant human), 0.33 µg/mL hydrocortisone, 0.39 µg/mL epinephrine, 10 µg/mL transferrin (recombinant human), 0.05 mM CaCl₂ (all from PromoCell) and 30 µg/mL gentamicin sulphate (Serva, Heidelberg, Germany). For experiments under high calcium conditions, the cells were grown in KGM2 with 2 mM CaCl₂. The human epithelial cell line T84 was maintained at 37 °C in a humidified incubator under 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium (1:1), supplemented with 20% heat-inactivated fetal bovine serum, 50 U/mL streptomycin and 50 U/mL penicillin.