Figure 6

Evaluation of redox homeostasis. (a,b) Flow cytometry analysis of intracellular ROS levels after incubation of LoVo with 10 µM DCFH-DA was performed as described under Method section. (c) Time-line (12, 24, 48, and 72 h) of extracellular H₂O₂ production was determined with Amplex Red H₂O₂/peroxidase assay. Data are shown as mean ± SD of three independent experiments. (d) Time-dependent mitochondrial superoxide levels assessed in LoVo cells by MitoSOX-confocal microscopy analysis after 12, 24, 48, and 72 h of treatment with milk (40% v/v), δVB (2 mM), milk + δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). (e) Representative confocal images of MitoSOX (red) and phalloidin (green) in control cells (Ctr), milk (40% v/v), δVB (2 mM) or milk + δVB for 72 h. Nuclei were counterstained with DAPI. Scale bar, 50μm. (f,g) MitoSOX-based flow cytometry in cells treated with vehicle (Ctr), milk (40% v/v), δVB (2 mM) or milk + δVB, for 72 h in serum-free medium. Results are expressed as mean fluorescence intensity (MFI). (h,i) Percentage of apoptosis and representative dot plots of annexin V-FITC and PI-stained cells analyzed by flow cytometry in LoVo cells treated for 72 h with HBSS-10 mM Hepes (40% v/v) (Ctr) milk+ δVB, or NAC + milk+ δVB. Data are expressed as mean ± SD of n = 3 experiments. At least 10.000 events were acquired. (j) The effect of 72 h treatment with δVB (2 mM) on mitochondrial function was determined by assaying cytochrome oxidase activity in whole LoVo lysates. ^*P < 0.05 vs Ctr, **P < 0.01 vs Ctr, ^††P < 0.01 vs milk, °P < 0.01 vs δVB, ^§P < 0.001 vs Ctr; ^§§P < 0.0001 vs Ctr, ^¶P < 0.001 vs milk, ⁺P < 0.05 vs milk+δVB, ⁺⁺P < 0.01 vs milk+δVB.