Axonal density was calculated by dividing the number of axons in an individual image by the area

Ten l of sample were loaded on 96-well white plates, and luminescence was quantified in a microplate luminometer. Luminescence was then normalized with sample protein concentration as determined by Bradford assays. Western Blot Analysis For protein expression analysis, cells were seeded on 100 mm plates. After treatments, cells were lysed with ice-cold lysis buffer. Thirty micrograms of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Blots were incubated overnight at 4C with appropriate antibodies: hydroxylated-HIF- 1, phosphorylated AKT and phosphorylated mTOR as well as glyceraldehyde3-phosphate dehydrogenase as a loading control. Immunoreactive polypeptides were visualized using horseradish peroxidase conjugated secondary antibodies and enhanced-chemiluminescence detection reagents following manufacturersupplied protocols. Inmunoblots were analyzed by means of Un-Scan-It Gel PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19722344 6.0 to provide quantitative values for relative expression of each protein. 4 / 15 Melatonin Regulation of Warburg Effect in Cancer Cells 5 / 15 Melatonin Regulation of Warburg Effect in Cancer Cells Fig 1. Ewing sarcoma cells exhibit the main hallmarks of Warburg effect. The following parameters indicative of aerobic glycolysis were determined in TC-71 and sw-1353 cell lines: Glucose uptake; Intracellular lactate levels; LDH activity, normalized versus protein content; ATP levels; and Mitochondrial membrane potential was expressed as RFUs. TC-71 cells were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1972496 treated with or without 16.2 mM oxamate for 48 hours, and cell survival rate was determined by means of trypan blue staining. Results were expressed as a percentage of death cells. p0.05 vs. vehicle-treated cells. doi:10.1371/journal.pone.0135420.g001 Data analysis Results are represented as the average value of at least three independent experiments. Data are represented as the mean +/- SEM. Significance was tested by one-way ANOVA followed by a Student-Newman-Keuls multiple range test. Statistical significance was accepted when P 0.05. Results Ewing sarcoma cells but not chondrosarcoma cells exhibit features of Warburg effect Most cancer cells frequently display a metabolic profile different to normal cells. They have higher rates of cytosolic glycolysis, uptaking more glucose and producing an excess of lactic acid. We evaluated these parameters characteristic of the tumoral Warburg effect in two sarcoma cell lines: sw-1353 chondrosarcoma cells and TC-71 Ewing sarcoma cells. As shown in Fig 1A, TC-71 Ewing sarcoma cells consumed higher levels of glucose than sw-1353 chondrosarcoma cells. This is accompanied by an increased lactate production and lactate dehydrogenase activity, suggesting an increase in fermentative metabolism in TC71 cells in comparison to sw-1353 cells. TC-71 cells also exhibited lower levels of ATP and a less significant mitochondrial membrane potential than sw-1353 cells. Sutezolid Inhibition of LDH by oxamate in the TC-71 cell line caused cell death, indicating that this metabolic pathway is essential for their survival. All results together show an evident difference in basal energy metabolism between chondrosarcoma and Ewing sarcoma cells, which in turn may be related to the differential effect of melatonin on both cell types. In those cells where we found Warburg effect melatonin induces cell death, whereas in those cells that present “normal” glycolytic metabolism with little or no participation of the aerobic glyco