Research Paper Volume 4, Issue 1 pp 28—39

Figure 3. Flow cytometry analysis of p53^−/− HCT116 cells transfected with either EGFP-p53R⁷² or EGFP-p53P⁷² pCMS plasmid. After 24 hours from transfection, cells were treated with 100 nM rotenone and incubated for additional 24 hours, then stained for 8-oxo-dG or TMRM (see materials and methods). (A) transfection efficiency. Cells contained in R1 are considered EGFP-positive. Ctr: non-transfected cells. (B) 8-oxo-dG detection. The cells in (R1) and (not R1) are evaluated for 8-oxo-dG fluorescence (RPE). Numbers represent the percentage of cells with high 8-oxo-dG fluorescence and are expressed as mean ± st. dev. of three independent experiments. As showed EGFP-negative cells (not R1, not expressing p53) are almost all positive for 8-oxo-dG fluorescence (more than 90%), while when considering EGFP-positive cells (R1, expressing p53), the cells that were transfected with p53R⁷² which resulted positive for 8-oxo-dG decreased to 69%, with respect to 92% of those that were transfected with p53P⁷². See text for comment. (C) Mitochondrial membrane potential (MMP) analysis. The cells in (R1) were evaluated for MMP by using the potentiometric dye TMRM. Black line: control cells; red line: rotenone treatment. Decrease in MMP was expressed as the difference (Δ) of TMRM fluorescence intensity between rotenone-treated and control cells. Cells positive for EGFP-p53R⁷² (left panel) display a lower decrease in MMP with respect to those positive for EGFP-p53P⁷² (right panel). The graphic shows data related to 3 independent experiments (mean ± st. dev.) * Student' t test p= 0.022. NT, not transfected.