Dulbecco modified eagle medium (dmem)

CAA was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, U.S.A.) to a concentration of 200 μM and stored at −20°C as a stock solution. The stock solution was diluted with Dulbecco’s modified Eagle’s medium (DMEM, HyClone, U.S.A) to a final concentration of 50 μM before use. Dexamethasone (Cell Signaling Technology, #9668) was diluted with DMEM to a final concentration of 1 μM. DMSO was added in the vehicle control group. TGF-β (240-B; R&D Systems, MN, U.S.A.) was diluted to a concentration of 6.4 ng/ml, and IL-6 (206-IL/CF, R&D Systems, MN, U.S.A.) was diluted to a concentration of 8 ng/ml before use. Insulin (Cell Signaling Technology, #9668), human IL-6 high sensitivity enzyme-linked immunosorbent assay (ELISA) Kit (Abcam, ab46042, U.S.A), human IL-17 High Sensitivity ELISA Kit (Abcam, ab46042, U.S.A), propidium iodide (Cell Signaling Technology, #9668), 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, #9668), primary antibodies including anti-IL-17RC, anti-IL-6, anti-p-IR (Cell Signaling Technology, #9542), anti-GLUT1 (Cell Signaling Technology, #9542), and monoclonal mouse anti-GAPDH (Santa Cruz, CA, U.S.A.), and horseradish peroxidase (HRP)–conjugated polyclonal goat anti-mouse and anti-rabbit (both from Santa Cruz, CA, U.S.A.) were used in the present study. Human IL-17RC adenovirus was purchased from Vector Biolabs (Malvern, PA, U.S.A.).

Primary cultures of scleral fibroblasts were obtained from whole sclera explants of 2-week-old guinea pigs. The scleral tissue was cut into tissue blocks of 1 × 1 × 1 mm³ under sterile conditions and carefully placed into separate flasks in Dulbecco's modified Eagle medium (DMEM; High Glucose, Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). Fibroblast cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂ until confluent. The medium was replenished twice a week. Cells at 80% confluence were passaged by using 0.25% trypsin-EDTA (Gibco, USA). The cells were identified by vimentin detection, as previously described [33 (link)]. Fibroblasts between 3 and 5 passages were used for the experiments. Cells were washed and cultured with DMEM (High Glucose) without FBS for 24 hours and then incubated with basal medium plus 0.1 μM tunicamycin (TM; Cell signaling technology, USA), 2.5 mM 4-phenylbutyric acid (4-PBA; Sigma-Aldrich, USA), and with 0.1 μM TM + 2.5 mM 4-PBA to extract proteins and total RNA, respectively.