2.7. Assay of cellular TrxR activity

TRFS-green is a specific TrxR probe by our previously established⁴². HeLa cells (2 × 10⁵) were plated into a 12-well plate and allowed to adhere. The HeLa cells were incubated with 0, 10, 20, or 40 µM of ONP or 40 µM C2 or C3 for 8 h. Then the cells were removed from the medium and subsequently treated with TRFS-green (10 µM) in a fresh FBS-free medium for 4 h at 37 °C in dark. The cells were washed to remove the residual TRFS-green. The cells were imaged under a fluorescent microscope (Floid Cell Imaging Station, Thermo Fisher).

Fast-TRFS-based cell lysate assay is the rapid detection of TrxR activity in crude protein as the source of TrxR by our previously described method⁴³. Briefly, HeLa cells were plated in 100-mm culture dishes and were cultured overnight. The cells then were collected and lysed with RIPA buffer. Total cellular protein contents as the source of TrxR were quantified by the Bradford procedure. Subsequently, the HeLa cell lysate (0.3 mg·mL⁻¹) was incubated with NADPH (100 µM) for 5 min at 37 °C. Test drug, ONP (10, 20, or 40 µM each), and blank sample (0.1%DMSO) were then added and the mixture was continued to incubate for 1 h. The probe Fast-TRFS (10 µM) and NADPH (100 µM) were added to initiate the enzymatic reduction of Fast-TRFS. The fluorescence change at 460 nm was recorded (λex = 345 nm) for 10 min on a fluorescent plate reader (Tecan Infinite M200), and the rate of fluorescence increase within the initial 5 min was calculated. The relative TrxR activity was expressed as the percentage of the DMSO-treated sample.

HeLa cells were treated with different concentrations of ONP, C2, and C3 for 12 or 24 h. The cells were washed and harvested. Subsequently, the total cellular proteins were extracted and quantified by the RIPA buffer and the Bradford procedure, respectively. The intracellular TrxR activity was measured by the endpoint insulin reduction assay described as our published procedures^30,37.