Thioredoxin Reductase Inhibition Assay

TrxR1 activity was assessed using the Thioredoxin Reductase Assay Kit according to the manufacturer’s instructions (Sigma-Aldrich, Milan, Italy). TrxR1 activity was spectrophotometrically assayed at 412 nm absorbance using a microplate reader (Tecan Trading AG, Switzerland). The assay is based on the NADPH-dependent reduction of the substrate, reacting with 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB). Different doses of Au3BC, Au4BC, and auranofin (positive control inhibitor) were incubated with TrxR1 extracted from rat liver. Furthermore, 10 μg of protein extracted from Huh7 cells was used for determination of TrxR1 activity in cell lysates.