Normal skin was dissected from the dorsum of the hand and palm of three fresh cadavers, donated for medical research and education to the Department of Anatomy, Chungnam National University School of Medicine. Tumor tissues were obtained from the biopsy specimens of three patients with squamous cell carcinoma (SCC) and three patients with syringoma. The tumor specimens and prepuce of the penis were obtained during dermatologic surgery in accordance with the guidelines of the ethics committee of Chungnam National University Hospital (IRB No. 2012-08-023).

To generate SV-40T-transformed human epidermal keratinocytes (SV-HEKs), primary epidermal keratinocytes were cultured according to a previous method23. In SV40 transformation, the retroviral vector pLXIN-SV40T was stably transfected into PT67 cells (Clontech Laboratories, Mountain View, CA, USA), a recombinant retrovirus-packaging cell line. Retrovirus-containing medium was collected, filtered through a 0.22-µm low-protein-binding filter (Millipore, Billerica, MA, USA), and transferred to primary cultured keratinocytes. Following overnight infection, the retrovirus-containing medium was replaced with fresh medium and the cells were incubated for 2 days. Transfectants were selected in medium containing G418 (Sigma-Aldrich, St. Louis, MO, USA) (1 mg/ml) for 4 weeks, as reported previously23. SV-HEKs were maintained in keratinocyte serum-free medium supplemented with bovine pituitary extract and recombinant human epidermal growth factor (Life Technologies Corporation, Grand Island, NY, USA). SCC12 cells were purchased from Invitrogen (Carlsbad, CA, USA). The SCC12 cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Life Technologies Corporation).

Cellular proteins were separated using SDS-PAGE, transferred to nitrocellulose membranes, and incubated with appropriate antibodies overnight at 4℃ with gentle agitation. The blots were incubated with peroxidase-conjugated secondary antibodies for 2 h at room temperature, and the signals were visualized using enhanced chemiluminescence (Intron, Daejeon, Korea). We used primary antibodies against PAR-2, phosphorylated-ERK (p-ERK), filaggrin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and loricrin (Covance Research Products, Denver, PA, USA).

Paraffin-embedded tissue was cut into 4-µm sections and mounted on slides. The tissue sections were deparaffinized and antigen retrieval was performed by heating the slides for 4 min in 10 mmol L^-1 citrate buffer (pH 6.0) in a pressure cooker. Subsequent procedures were conducted at room temperature. The sections were pretreated with 1% H₂O₂ in methanol for 30 min to quench endogenous peroxidase activity. The tissue sections were treated with PAR-2 antibody (Santa Cruz Biotechnologies) for 1 h followed by treatment with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was visualized by incubation for 1 h with an avidin-biotin-peroxidase complex (Vectastain ABC system; Vector Laboratories) in phosphate-buffered saline (PBS) and for 5~10 min in 0.05% 3,3'-diaminobenzidine, 0.01% H₂O₂ in 0.1 mol L^-1 PBS. The immunostained tissue slides were counterstained with 0.1% methyl green. The immunolabeled sections were dehydrated though graded ethanol solutions, cleared in xylene, and mounted. Negative control sections were treated as described above, but the primary antibody was omitted.