Epilife

Human primary KCs were collected from the foreskins of twelve patients (aged 8 to 30 years) who underwent urological surgery at the Department of Urology, Xijing Hospital, as previously described 33 (link). Keratinocyte complete media (EpiLife, Thermo Fisher Scientific, USA) supplemented with EpiLife medium +60 µM calcium/EpiLife Defined Growth Supplement (EDGS, Thermo Fisher Scientific, USA) was used to sustain human primary KCs. Penicillin‒streptomycin (50 U/mL) was added. Human primary KCs were then treated with ENO1 siRNA, ENOBlock, BI-D1870, K17 and mutant K17 overexpression plasmids in 6-well plates.
The human HaCaT keratinocyte cell line was purchased from KeyGEN Biotech (GB300, Nanjing, China), cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco-Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-Invitrogen, Carlsbad, USA) and maintained at 37°C in a humidified environment with 5% CO₂. After 24 h of starvation, cells at 40-60% confluence were stimulated with IL-17, IL-22, tumor necrosis factor-α (TNF-α), IL-1α and oncostatin M (OSM) (50, 20, 50, 20 and 20 ng/mL, respectively) (PeproTech, Rocky Hill, NJ, USA) for 48 h, and PBS treatment served as a negative control.

Epidermal equivalents were generated by seeding 1 × 10⁶ keratinocytes onto polyethylene terephthalate hanging cell‐culture inserts with pore size of 0·4 μm (Merck, Gillingham, UK; MCHT12H48), coated with human type I collagen (ThermoFisher Scientific, R011K), and cultured in low‐calcium EpiLife for 48 h. Equivalents were then grown at the air/liquid interface in high‐calcium EpiLife supplemented with vitamin C (50 μg mL^–1) for 7 days, replacing medium every 48 h. Skin equivalents were fixed in 4% (w/v) paraformaldehyde, embedded in paraffin and processed for immunohistochemistry or haematoxylin and eosin staining. Cell proliferation was assessed by counting nuclei in 500‐μm fields of view.

The hair shaft and hair bulb regions of the HFs were cut off to prevent contamination with other cells. The ORS cells were cultured as described before (Kwack et al., 2008 (link)). Trimmed hair follicles were immersed in Dulbecco’s modified eagle medium supplemented with 20% fetal bovine serum in Biocoat collagen type I-coated tissue culture dishes (Corning, Kennebunk, ME, United States). On the third day of culture, the medium was changed to EpiLife (Gibco-BRL, Gaithersburg, MD, United States) containing 1% antibiotic-antimycotic solution and 1% EpiLife defined growth supplement. Once subconfluent, the cells were harvested with 0.25% trypsin/10 mM EDTA in phosphate-buffered saline (PBS), split at a 1:5 ratio, and maintained in EpiLife medium. ORS cells in passages 1–4 were used in this study.