Aspirin

OVA (grade V), spermine, diclofenac, and meloxicam were purchased from Sigma-Aldrich (St Louis, MO, USA). Aspirin and indomethacin were obtained from Wako Pure Chemicals (Osaka, Japan) and Nacalai Tesque (Kyoto, Japan), respectively. Alum adjuvant (Imject^® Alum) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Horseradish peroxidase (HRP)-conjugated mouse anti-rat IgE (MARE-1) and HRP-conjugated goat anti-rat IgG₁ were purchased from GeneTex (Irvine, CA, USA) and Bethyl Laboratories (Montgomery, TX, USA), respectively. All chemicals used were of the highest purity available.

Two PROS fibroblast cell lines were established from the patient's lower limb and perineum. Two control fibroblast cell lines (C2 and C3), which were previously established from the skins of two unrelated healthy volunteers of a 36-year-old male and a 27-year-old female, respectively, [62 (link)] were used. Additionally, a normal human dermal fibroblast cell line established from the skin of a 14-year-old male (NHDF-c, c-12300) was purchased from PromoCell GmbH (Heidelberg, Germany). Fibroblast cells were cultured as previously described [62 (link)]. Briefly, fibroblasts were maintained in Eagle's minimal essential medium (EMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS, penicillin (100 U/mL), streptomycin (0.1 mg/mL), L-glutamine (1.75 mM), HEPES (10 mM), and NaHCO₃ (0.13%). All supplemental reagents, except for FCS, were purchased from Nacalai Tesque (Kyoto, Japan). The cells were plated on 35-mm collagen-coated dishes at a density of 1.1 × 10⁴ cells/cm² for biochemical experiments. Fibroblast cells were treated with rapamycin (LKT Laboratories, St. Paul, MN, USA), NVP-BEZ235 (Chemscene, Monmouth Junction, NJ, USA), aspirin (acetylsalicylic acid; Wako, Osaka, Japan) and metformin hydrochloride (Sigma-Aldrich) for 2 days, washed with PBS, and then harvested and subjected to SDS-PAGE and western blotting.