Catalytically active TRX is required to maintain tRNA-LC activity during oxidative stress and for its reactivation after oxidative stress levels decrease. (A) ShRNA-resistant wild-type FLAG-TRX CxxC (labeled as CxxC) or active site double mutant FLAG-TRX SxxS (labeled as SxxS) were constitutively overexpressed in shTRX cells. Silencing of endogenous TRX was induced by the addition of 2 µg/mL Dox for 3 d. Cells were treated with menadione for 1 h before harvest and lysis, and RNA ligation activity was assayed by incubating cell lysates with a double-stranded, radiolabeled RNA substrate containing a 3′ phosphate on one strand and a 5′OH on the complementary strand. Products of the reaction were resolved by denaturing urea polyacrylamide gel electrophoresis and visualized by phosphorimaging. (B) Quantification of band intensities from A. The substrate conversion rate was calculated as the quotient of the substrate and the sum of substrate and product band intensities and normalized to untreated shCtrl sample (n = 3, mean values ± SEM are shown. Significances were analyzed using unpaired Student's t-test assuming unequal variances, with [*] P < 0.05; [**] P < 0.01; [***] P < 0.005). Top left graph: comparison of ligation product in shCtrl versus shTRX FLAG-TRX CxxC cells. Top right graph: comparison of ligation product in shCtrl versus shTRX FLAG-TRX SxxS cells. Bottom left graph: comparison of ligation product in shTRX versus shTRX FLAG-TRX CxxC cells. Bottom right graph: comparison of ligation product in shTRX versus shTRX FLAG-TRX SxxS cells. (C) HeLa cells were transfected with siRNA pools targeting TXN mRNA (siTRX) or a control siRNA (siCtrl) for 3 d. Cells were treated with 30 µM menadione for 30 min, washed, and allowed to recover for 0, 1, or 3 h in medium containing 10 µg/mL CHX. As a control, cells were either left untreated or treated with CHX alone for 5 h. Cells were lysed and assayed for RNA ligation as in A. (D) HeLa cells were transfected with siRNA pools targeting TXN mRNA (siTRX) or a control siRNA (siCtrl) for 3 d. Cells were treated with 125 µM H2O2 for 30 min, washed, and allowed to recover for 0, 1, or 3 h in medium containing 10 µg/mL CHX. As a control, cells were either left untreated or treated with CHX alone for 5 h. Cells were lysed and assayed for RNA ligation as in A.
