Biochemical and Biophysical Research Communications
Modification of glycolysis affects cell sensitivity to apoptosis induced by oxidative stress and mediated by mitochondria☆
Section snippets
Materials and methods
Cell culture and viability assay. The dihydrofolate reductase (dhfr)-deficient CHO (CHO dhfr−) cells were maintained under a humidified atmosphere of 5% CO2 at 37 °C in NaHCO3-buffered F-12 medium supplemented with l-glutamine, 10% fetal bovine serum, penicillin (1000 U/ml), and streptomycin (1000 μg/ml). For determination of cell viability, cells (3 × 105) were seeded onto 60-mm dishes, cultured for 12 h, and then incubated for an additional 12 h in fresh medium before exposure to tert-butyl
Effect of modification of the glycolytic pathway on the input of respiratory substrate into mitochondria
In this study, we tried to bypass the glycolytic pathway by overexpressing cGPDH and anti-sense LDH-A RNA. To maximize the effect of anti-sense ldh gene in the cell, we used dihydroforate reductase(dhfr)-deficient CHO cells (CHO dhfr−), which are available for an efficient co-amplification system using dhfr gene in the presence of an amplificable marker, methotrexate (MTX). One of the several target proteins to amplify the gene in mammalian cells is DHFR, an enzyme that catabolizes the
Acknowledgements
This work was supported by a grant from BioGreen 21 Program, Rural Development Administration, Republic of Korea.
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Abbreviations: ROS, reactive oxygen species; cGPDH, cytosolic glycerol-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; t-BOOH, tert-butyl hydroperoxide; PBS, phosphate-buffered saline; MPT, mitochondrial permeability transition; ΔΨm, mitochondrial membrane potential; Rh123, rhodamine 123; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; NEM, N-ethylmaleimide.
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These authors contributed equally to this work.