Nheks

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.

Cell lines 293FT, A549, 1321 N1, Shh-Light2 (Shh-L2) and U2OS were purchased from ATCC. HUH7 cells were a kind gift from Dr. Pirjo Spuul. Adult pooled normal human epithelial keratinocytes (NHEKs, PromoCell) (kind gift from Dr. Ana Rebane) were grown in Defined Keratinocyte-SFM Medium (DKSM) (Gibco, Thermo Fisher Scientific). 293FT, A549, 1321 N1, and Shh-L2 cell lines were cultured in Dulbecco's Modified Eagle Medium-high glycose (DMEM, Pan Biotech) supplemented with 10% foetal calf serum (FCS, Pan Biotech) and 1% penicillin/streptomycin (PEST, Sigma-Aldrich). The growth medium of Shh-L2 cells also contained 0.4 mg/ml G418 (Sigma-Aldrich) and 0.1 mg/ml zeocine (Invitrogen). U2OS cells were propagated in Iscove's Modified Dulbecco's Medium (IMDM, Pan Biotech), 10% FCS and 1% PEST. Cells were propagated at 37°C in 5% CO₂. HUH7 cells were transfected using Lipofectamine 2000 (Life Technologies). 293FT and Shh-L2 cells were transfected using polyethylenimine (PEI) as previously described [9 (link)]. U2OS cells were co-transfected with HPV minicircle genomes and GLI1FL or GLI1ΔN encoding constructs by electroporation (220 V and 975 μF) using a Gene Pulser XCell system (Bio-Rad Laboratories).

NHDFs and NHEKs were purchased from Promocell, Tebu—bio, GIBCO or Cambrex. HMECs were purchased from Bio-Whittaker. For details, see Supplementary Table 1. Cells were grown at 37 °C in an atmosphere of 5% CO₂ and at the atmospheric O₂ tension. NHEKs were cultured in the KGM-Gold^TM bulletkit medium (Clonetics). It consists of modified MCBD153 with 0.15 mM calcium, supplemented with bovine pituitary extract, epidermal growth factor, insulin, hydrocortisone, transferrin and epinephrine. Such a serum-free low-calcium medium has been shown to minimize keratinocyte terminal differentiation66 (link). NHDFs were cultured in FGMTM-2 bulletkit medium. HMECs were cultured in MEGM^TM bullekit medium.
Cells were seeded as recommended by the supplier and subcultured at 70% confluence. The number of PDs was calculated at each passage by using the following equation: PD=log (number of collected cells/number of plated cells)/log2.

The HFDPCs were purchased from PromoCell (Heidelberg, Germany) and cultured in Follicle Dermal Papilla Cell Growth Medium Kit (PromoCell) supplemented with 1% (v/v) penicillin/streptomycin (PS, Gibco BRL). Normal human epithelial keratinocytes (NHEKs) were purchased from PromoCell and cultured in keratinocyte growth medium 2 (PromoCell) supplemented with 1% (v/v) PS. The cells were incubated in 150-mm cell culture Petri dishes (Corning Inc., Corning, New York, NY, USA) until they reached 70–80% confluence. The cells were incubated at 37 °C and 5% CO₂ saturation. The cell culture medium was changed every 3 days. Cells within 4 passages were used for the experiments.