Figure 2.

NAC pretreatment decreases levels of reactive oxygen species and increases GSH under oxidative conditions in H9c2 cells. (A) H9c2 cells were incubated with CM-H2DCFDA dye at a concentration of 10 µM for 30 min at 37°C. Cells were pretreated with 4 mM NAC for 1 h and were then treated with 0.75 mM H₂O₂ for the indicated durations (15 and 30 min). Fluorescence was quantified using flow cytometry with excitation and emission wavelengths of 485 and 530 nm, respectively. Values are presented as the means ± standard deviation (n=6). Intracellular (B) total and (C) reduced GSH levels were determined using a GSH reductase recycling assay. (D) Immunofluorescence of mito-roGFP (green) in H9c2 cells (magnification, x400). Scale bar, 25 µm. (E) Western blotting revealed the expression levels of mito-roGFP and β-actin. (F and G) H9c2 cells were transfected with mito-roGFP or vector for 48 h and were then treated with 0.75 mM H₂O₂ for the indicated times (15 and 30 min). Non-reducing western blotting was performed to detect the redox state of mito-roGFP. Data are presented as the means ± standard deviation (n=6). ^*P<0.05, ^**P<0.01, ^***P<0.001. GFP, green fluorescent protein; GSH, glutathione; H₂O₂, hydrogen peroxide; mito-roGFP, mitochondrial redox-sensitive GFP; NAC, N-acetylcysteine.