Psoriasis-like Keratinocyte Stimulation

HaCaT and HEKa cells (human keratinocyte cell lines) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂. Cells were stimulated with 10 ng/ml M5 (IL-1α, IL-17A, IL-22, oncostatin M, and TNF-α; PeproTech, USA) for 24 hours at 37°C to mimic a psoriasis-like environment in keratinocytes [17 (link)–20 (link)]. Cells were then either left untreated or pretreated with 1 μg/ml C23 (GRGFSRGGGDRGYGG synthesized from GenScript, Piscataway, NJ; dissolved in phosphate-buffered saline) for 30 minutes and then were stimulated with 1 μg/ml rhCIRP (APG886Hu01, Cloud-Clone Corp, Texas, USA) for 1 hour. After rhCIRP stimulation, cells were collected for qRT-PCR and western blot analysis.