Fig 1: TrxR1 and TRP14 counteract intracellular protein persulfide accumulation.S3 refers to sampling according to Fig. 1. (A) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected HEK293 cells, compared to control cells. GAPDH is applied as a loading control. (B) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. (C) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (*P < 0.05 using the paired t test); 100% in the control cells corresponds to 1.52 ± 0.55 µg/mg total protein. Error bars represent SDs of n = 4 experiments. (D) Two hours of treatment with 200 µM polysulfide induced higher levels of intracellular protein persulfides in intact TrxR1 and TRP14 knockdown cells compared to control cells. (E) A modified SRB cytotoxicity assay revealed that control cells (•) are significantly more viable upon polysulfide exposure than TrxR1 knockdown (?) cells (***P < 0.0001 at 0.5 and 1 mM polysulfide concentration). Data points and error bars represent the average and SD, respectively, of n = 4 independent experiments of triplicate measurements.