Research Paper Volume 2, Issue 8 pp 487—503

Figure 2. (A) a Intracellular redox state under nutrient restriction. a pseudocolor image (color bar on the left) of Phoenix cells expressing a redox-sensitive variant of the Yellow Fluorescent Protein (rxYFP) after 24 hours incubation in the absence of glucose (upper) and 30 minutes glucose re-feeding, in serum-free medium. Color shift from red to yellow indicates reduction of the fluorescent sensor. b Quantitation of mean R values over several regions of interest is reported. Data sets were compared by two-tailed t-test for independent samples. c Microfluorimetric analysis of reduced intracellular reduced NAD(P)H, based on cell green autofluorescence. Cells were excited in the two-photon mode at 366 nm and autofluorescence collected between 380 and 550 nm. Increase in cell brightness in the glucose-fed samples indicates accumulation of reduced pyridine nucleotides. (B) a Effect of antioxidants Catalase and N-Acetyl-Cysteine on cell viability in the presence and absence of glucose. Cells were transfected with a construct encoding human Catalase or the corresponding empty vector 48 hours before nutrient and serum starvation. Mock-transfected cells were also treated with 10 mM NAC as an alternative ROS scavenger. Values are mean±SD of triplicate wells. The experiment was repeated twice with identical results. b Cytofluorimetric analysis of cells loaded with the redox sensitive die H2-DCF-DA and exposed to a bolus (1 mM) of extracellular Hydrogen Peroxide. Decreased oxidation in the Catalase-transfected samples confirms elevated H2O2 degrading capacity in these cells. (C) Lack of effect of the longevity protein Sirt1 and the mitochondrial superoxide scavenger SOD2 on Phoenix cell viability in the presence of glucose and under glucose deprivation. Cell viability was scored at 72 hours after cell starvation. Representative of two comparable experiments. (D) Western blot analysis of Sirt1 (a) and SOD2 (b) expression in transfected cells. Transfection efficiency was normally around 50% based on expression of GFP.