Fig. 5

Role of NQO1 overexpression in oxidative stress in HG-cultured HK-2 cells. A The protein levels of NQO1, Sirt1, Nox1 and Nox4 were analyzed by western blotting (n = 3). B The mRNA levels of NQO1 and Sirt1 were measured by RT-qPCR (n = 3). C Sirt1 activity in HK-2 cells (n = 6). D The intracellular NAD+/NADH ratio was measured in HK-2 cells (n = 6). E–I The expression levels of NQO1, Sirt1, Nox1 and Nox4 were measured by immunofluorescence (scale bar, 50 μm, n = 9). E Mitochondrial ROS was assessed by the fluorescence probe MitoSOX Red (scale bar, 10 μm, n = 6). J Quantitative analysis of mitochondrial ROS. K Intracellular ROS were measured by flow cytometry and quantitative analysis (n = 6). NG: 5.6 mM d‐glucose, M: NG + mannitol (24.4 mM), HG: 30 mM d‐glucose, HG + C: HG + control pcDNA3.1(+), HG + NQO1 O/E: HG + NQO1 pcDNA3.1(+). Values are expressed as the mean ± SD. **P < 0.01 versus NG group; ^#P < 0.05, ^##P < 0.01 versus HG + C group