Fig. 7: Glutathione reductase deletion protects against H₂O₂-induced NADP oxidation in HEK293 cells.

a Cartoon showing two possible pathways by which electrons from NADPH can be transferred to H₂O₂ in the mammalian cell cytosol, GSR glutathione reductase, TPX thioredoxin peroxidase, TRX thioredoxin, TXNRD1 thioredoxin reductase. The ‘?’ indicates a lack of information concerning the relative importance of glutathione as a reductant for thiol peroxidases in the mammalian cell cytosol. Created with BioRender.com. Response of NAPstar3b in the cytosol of wild-type and glutathione reductase deleted (GSR KO) cells to 100 µM diamide (b) (n = 17 or 18 individual cells for wild-type and GSR KO, respectively, measured in two separate experimental repeats) or 500 µM diamide (c) (n = 22 or 19 individual cells for wild-type and GSR KO, respectively, measured in two separate experimental repeats. d Box and whisker plot showing the change in TS/mC after treatments in b and c. Response of NAPstar3b expressed in the cytosol of wild-type and GSR KO cells to 50 µM H₂O₂ (e) (n = 103 or 46 individual cells for wild-type and GSR KO respectively, measured in four and three separate experimental repeats) or 100 µM H₂O₂ (f) (n = 98 or 76 individual cells for wild-type and GSR KO, respectively, measured in four and five separate experimental repeats. g Box and whisker plot showing the change in TS/mC after treatments in e and f. Response of NAPstar3b in the cytosol of HEK293 cells, treated either with a vehicle control, the thioredoxin reductase inhibitor auranofin (h) or the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)−1-nitrosourea (BCNU) (i) (n = 85, 97, 79 individual cells for control, auranofin and BCNU treatments respectively, monitored in the course of 3 experimental repeats). j Box and whisker plot showing the change in TS/mC after treatments in h and i. Data were normalised to the average value at the beginning of the measurement. For all box and whisker plots, boxes show the interquartile range, with the middle line defining the median. ‘X’, represents the mean values. Whiskers show the minimum and maximum values, excluding outliers. Dots indicate outlier values, which are defined as being 1.5 times the interquartile range above and below the third and first quartile respectively. P-values in panels d. and g, were determined by unpaired two-tailed Student’s t-tests. P-values in panel j. were determined by a one-sided ANOVA test.