Figure 9. Generation and measurement of nuclear-localized H₂O₂ and oxidation events using the NLS-HyPer-DAAO construct in human chondrocytes.

(A) An adenoviral vector encoding NLS-HyPer-DAAO was transduced into human articular chondrocytes and activated with varying concentrations of D-alanine (2-10 mM) for 15 mins to generate nuclear H₂O₂. Representative fluorescent images of the response to 6mM D-ala from n=3 independent samples. Images are shown in two different colors for presentation purposes. (B) Plot of calculated ratios between fluorescence intensities recorded using FITC and CFP excitation filters in response to varying concentrations of D-ala. Data was normalized to the average value prior to the addition of D-ala (representative data from n=3 independent samples). (C) Chondrocytes were treated with 6 mM D-ala for 0-60 mins prior to incubation in an NEM alkylating buffer to alkylate reduced thiols and cells were lysed in the presence of NEM. Under non-reducing conditions, immunoblotting for Prx1, Prx2 and Prx3 allowed for identification of Prx reduced monomers (labelled M on blots) and oxidized dimers (labelled D on blots). β-tubulin served as a loading control. (D) Densitometric analysis of D-alanine-induced Prx1, Prx2 and Prx3 dimer formation observed over the 60-min time course. Individual data points from n=5 independent samples are shown. Asterisks represent significant differences compared to untreated controls (*, p<0.05; **; p<0.01; ***; p<0.001) (Two-way ANOVA). (E) Human chondrocytes were co-transduced with ad-NLS-HyPer-DAAO and ad-Null or ad-SIRT6 vectors for 48 hrs. Plot of calculated ratios between fluorescence intensities recorded using FITC and CFP excitation filters in response to 6 mM D-alanine. Data was normalized to the average value prior to the addition of D-ala (representative data from n=3 independent samples).