Lipid extraction

The entire tissue of each RHEK model was removed from the membrane and was floated on trypsin (2.5 mg/mL) /EDTA (0.25 mg/mL) aqueous solution for 15 min at 37°C in a 5% CO₂ atmosphere for the SC separation. After adding the FBS, the SC separation was then performed using a microscope. The Obtained SC were washed with PBS and stored at -80°C until the determination of ceramides. Bligh-Dyer method described in previous studies [17, 38] was used for lipid extraction. Briefly, homogenization of SC was carried out using an ultrasonic homogenizer (AGC Techno Glass Co., Ltd. Shizuoka, Japan) in a mixture of chloroform, methanol and PBS (1:2:0.8). The mixture was then centrifuged (840×g, 15 min) after which the supernatants were obtained. Subsequently, the same volume of chloroform and PBS were added to each supernatant, solution were stirred using a vortex mixer for 20 min. After stirring, the solution was centrifuged (840×g, 15 min) and the bottom layer was obtained using a glass syringe. The obtained layer was dried at 30°C by N₂ gas. Remaining precipitates were used for quantification of total protein contents to correct the ceramide contents.