Measurement of GSH/GSSG

The cell’s capacity to generate reduced glutathione (GSH) and oxidized glutathione (GSSG) was quantified using the GSH/GSSG-Glo^TM assay (Promega Corporation, Madison, WI). Briefly, HEK293T and HepG3 cells were cultured in a 96-well clear bottom white plate at a density of 1 X 10⁴ cells/well and 2.5 X 10⁴ cells/well, respectively, and incubated ON for cells to attach. Following day, cells were treated with medium only and 100 μM NRH for 1, 4, and 24 h. After 24 h, cells were counted on a Celigo S imaging instrument to normalize for variations in cell density. Next, medium alone or medium containing NRH was removed, and the assay was performed according to the manufacturer’s protocol. Briefly, 50 μl of total glutathione lysis reagent or oxidized glutathione lysis reagent was added per well to immediately lyse the cells by continuously shaking the plate for 5 min at room temperature. Followed by the addition of 50 μl of luciferin generation reagent per well, the plate was briefly shaken and then incubated at room temperature for 30 min. Finally, 100 μl of luciferin detection reagent was added to the above mixture and incubated at room temperature for 15 min. Following assay incubation, luminescence was measured (in relative light units, RLU) on Infinite® M1000 PRO, TECAN microplate reader. Luminescence values were normalized to cell count, and the total glutathione and GSSG were measured. The GSH/GSSG ratio was calculated by subtracting two times normalized values of GSSG from the normalized total glutathione and divided this by normalized GSSG values (as shown in Eq 1).