Dulbecco s modified eagle s medium d mem nutrient mixture f 12 ham

We obtained elastic cartilage samples from microtia patients following the approved guidelines set by the ethical committee at Yokohama City University Graduate School of Medicine Hospital (approval no. B130905006). We stripped off the adipose tissue and microscopically separated the cartilage into three layers: the chondrium layer, interlayer, and perichondrium layer. The perichondrial sample was minced with scissors until almost no lumps of the cartilage matrix were found and the perichondrial cells were separated by shaking in a 0.2% collagenase solution (Worthington, Lakewood, NJ, USA) at 37 °C and 600 rpm for 2 h. The resulting suspension was filtered through a 40 µm cell strainer and centrifuged at 4 °C and 1500 rpm for 5 min to collect perichondrocytes. Having been suspended in growth medium and seeded on an uncoated 35 mm dish, the final concentration contained 10% fetal bovine serum (Biowest, Riverside, MO, US), Dulbecco’s Modified Eagle’s Medium (D-MEM)/Nutrient Mixture F-12 Ham (1:1) (Sigma-Aldrich, St. Louis, MO, US), and stabilized antibiotic–antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, US). When the perichondrocytes adhered and became confluent, the cells were collected using 0.05% trypsin–EDTA (1×) (Thermo Fisher Scientific, Waltham, MA, US) and expanded in a 225 cm² flask (n = 4).

Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham, APAP, l-glutamine, l-ascorbic acid, anti-GAPDH antibody and Triton^TM X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagenase, insulin human recombinant and cycloheximide were purchased from Wako Pure Chemical Industries (Osaka, Japan). Soybean trypsin inhibitor powder, HEPES buffer (1 M) and Dynabeads^® Protein G were purchased from Thermo Fisher Scientific (Gibco^®; Waltham, MA, USA). 2-Mercaptoethanol, dexamethasone, Nonidet^® P-40 and polyoxyethylene sorbitan monolaurate were purchased from Nacalai tesque (Kyoto, Japan). MG132 (Z-Leu-Leu-Leu-H aldehyde) was purchased from Peptide Institute (Osaka, Japan). Proteasome substrates II, III and VI, and Fluorogenic and anti-rabbit CYP3A1 antibodies were purchased from Merck Millipore (Calbiochem^®; Darmstadt, Germany). Penicillin G potassium and streptomycin were purchased from Meiji Seika Pharma (Tokyo, Japan). Anti-mouse CYP3A1, anti-rabbit CHIP, anti-rabbit gp78-2, anti-rabbit Nrf2 and anti-mouse Ub antibodies were purchased from Santacruz Biotechnology (Dallas, Texas, USA). P450-Glo^TM CYP3A assay with luciferin-IPA was purchased from Promega (Fitchburg, WI, USA).