Fig. 6

Energy-related kinases, nucleotides, and oxidative stress in HeLa clones. A–D Quantification of nucleotide ratios in HeLa cells (two clones of each condition, solid and hatched bars). A ATP/ADP ratio. B ATP/AMP ratio. C GTP/GDP ratio. D GTP/GMP ratio. E Expression of energy-related kinases in cell signaling and metabolism. Left: Representative immunoblots of cell extracts of the four HeLa clones for AMP-activated protein kinase (AMPK) and its activating phosphorylation at αT172 (P-AMPK), acetyl-CoA carboxylase (ACC), and its inhibiting phosphorylation at S79 (P-ACC), mitochondrial adenylate kinase isoform 2 (AK2), and mitochondrial ubiquitous creatine kinase (umtCK). Tubulin α served as loading control. Right: Quantification of band intensity ratios. Data given as means ± SEM (n=3), *p< 0.05, **p< 0.01 relative to CTR; #p< 0.05, #p< 0.01 relative to WT. F–I Quantification of oxidative stress markers determined in HeLa cells harboring empty vector control (CTR) or expressing WT or mutant NDPK-D (BD, KD). F Cellular levels of reactive oxygen species (ROS) determined with the fluorophore CM-H2DCFDA. Data are means ± SEM (n=3). G Reduced protein thiols (SH); their decrease indicates protein oxidation. Data are means ± SEM (n=3). H Lipid hydroperoxides. Data are means ± SEM (n=3). I Total antioxidant capacity determined by Ferric Reducing Ability of Plasma (FRAP), proportional to the reducing power of electron-donating antioxidants. Data are means ± SEM (n=6). *p< 0.05, ***p< 0.005 relative to control/empty vector (CTR); ^###p< 0.005 relative to wild-type (WT). For clone abbreviations, see Fig. 1