Research Paper Volume 11, Issue 15 pp 5726—5743

Figure 5. Glucose suppression of ROS is conserved in mammalian cells. (A) Short term rapamycin treatment increased intracellular ROS levels. Mouse embryonic fibroblasts (NIH3T3) cultured in medium supplemented with 1g/L glucose were treated with 100nM rapamycin for 1, 2, 4, and 8 hours. ROS levels were measured by staining cells with 2,7′–dichlorofluorescin diacetate (DCFDA), followed by flow cytometry analysis. (B) High glucose suppressed rapamycin-induced ROS in mouse embryonic fibroblasts. Mouse embryonic fibroblasts (NIH3T3) cultured in medium supplemented with 8g/L glucose were treated with 100nM rapamycin for 1, 2, 4, and 8 hours. ROS levels were measured by staining cells with 2,7′–dichlorofluorescin diacetate (DCFDA), followed by flow cytometry analysis. (C) High glucose suppressed ROS-induced cell death in mouse embryonic fibroblasts. NIH3T3 cells maintained in low (1g/L), normal (4g/L) and high glucose (8g/L) were treated with indicated concentrations of ROS-generator paraquat for 24 hours. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay. Means of 3 independent experiments were plotted with error bars showing the standard deviation. P value by student’s t-test: *, P<0.05. (D) Apoptosis was not significantly repressed by glucose. NIH3T3 cells maintained in normal (4g/L) and high glucose (8g/L) were treated 500μM ROS generator paraquat for 24 hours to induce cell apoptosis. Cell viability was measured by Propidium iodide (PI) staining and apoptosis were detected by Annexin V-FITC followed by flow cytometry analysis.