Gkt136901

To investigate the origin of redox signals, various inhibitors such as sodium diethyldithiocarbamate, GKT136901 (Sigma‐Aldrich, MO, USA), Mito‐tempo (MedChemExpress, NJ, USA), FCCP, oligomycin, rotenone and antimycin A (Agilent, CA, USA), and Batimastat (Selleckchem, Seoul, South Korea) were used as inhibitors. All of the inhibitors were incubated for 24 h at 37 °C. Furthermore, the cells were treated with CPI‐613 (MedChemExpress, NJ, USA) for drug screening and incubated for 24 h at 37 °C. After treatment, the cells were washed with DPBS for EC detection within 30 s and cytotoxicity testing.

NADPH oxidase activity was measured both in retina and RPE/choroid homogenates by lucigenin-enhanced chemiluminescence, following routine protocols in our laboratory [43 (link)]. To confirm the source(s) of superoxide anion (O₂^•−), homogenate samples were preincubated for 5 min at 37 °C with the following inhibitors at 0.1 mmol/L: diphenyleneiodonium, DPI (inhibitor of flavoproteins; Sigma-Aldrich, Madrid, Spain); oxypurinol (inhibitor of xanthine oxidase; Sigma-Aldrich, Madrid, Spain); and rotenone (mitochondrial chain inhibitor of electron transport; Sigma-Aldrich, Madrid, Spain). Following the same protocol, the inhibitor of NOX1/4 (0.1 µmol/L GKT136901; Sigma-Aldrich, Madrid, Spain, 492000), specific NOX1 inhibitor (0.5 µmol/L ML171; Sigma-Aldrich, Madrid, Spain, 175226) and the pan-NADPH oxidase inhibitor (10 µmol/L VAS2870; Sigma-Aldrich, Madrid, Spain, 5340320001) were used to explore the relative contribution of each NOX isoform in O₂^•− production [35 (link)]. Hydrogen peroxide (H₂O₂) levels were measured in retina homogenates by Amplex^TM Red hydrogen peroxide/peroxidase assay kit (A22188, ThermoFisher Scientific, Invitrogen, Spain) following the manufacturer’s instructions. Absorbance readings were obtained in 96-well plates at 560 nm. All measurements referred to the samples’ protein content, and results were always expressed as relative to the control group.

NADPH oxidase activity was measured in cornea homogenates by lucigenin-enhanced chemiluminescence, following routine protocols in our laboratory [48 (link)]. Potential sources of superoxide anion (O₂^.−) production in corneal samples were discriminated at 37 °C after a 5-min preincubation with 0.1 mmol/L DPI, oxypurinol, or rotenone (respective inhibitors of flavoproteins, xanthine oxidase, and mitochondrial electron transport chain; Sigma-Aldrich, Madrid, Spain). Similar protocols were followed when using an inhibitor of NOX1/4 (0.1 µmol/L GKT136901; Sigma-Aldrich, 492,000), specific NOX1 inhibitor (0.5 µmol/L ML171; Sigma-Aldrich, 175,226), and the pan-NADPH oxidase inhibitor (10 µmol/L VAS2870; Sigma-Aldrich, 5,340,320,001) to determine the relative contribution of each NOX isoform in total O₂^.− production. All measurements were referred to the samples’ protein content, and results were always expressed as relative to those in the control group.