Figure 4
Redox imbalance in XP-C cells due to mitochondrial ROS production. (A) Superoxide generation was measured by flow cytometry using 1 × 105 cells incubated with 5 µM Antimycin A, 5 µM DPI and 5 µM MitoSOX®. (B) H2O2 production was followed spectrofluorometrically with 5 µM Amplex UltraRed®, in presence of 5 µM Antimycin A, 5 µM DPI. Rate of H2O2 production calculated after calibration performed with known H2O2 concentrations. (C) SOD activity was measured using the Abcam kit, following the manufacturer’s instructions. (D) GPx activity was measured using 100 µg of total cellular protein. Oxidation of NADPH to NADP+ was monitored at 340 nm on a temperature-controlled spectrophotometer, at 25 °C. The results presented are the mean ± SD of n = 3 for superoxide production, n = 4 for H2O2 production, n = 3 for SOD and n = 3 for GPx activity. *p < 0.05 and **p < 0.01.
