Fig. 2.

Glutathione oxidation and ROS generation in response to AA and DHA treatments in MDA-MB-231 cells. (A) GSH and GSSG quantities were assessed in non-treated cells and cells treated with 2.5 or 10 mM AA or DHA for ½ hour and 2 h. Treatment with 250 μM diamide for 10 min was used as a positive control (PC). GSH/GSSG ratio value in non-treated cells is set as 1 and relative GSH/GSSG ratios calculated in treated conditions are represented as means ± SD of 4 independent experiments. Statistical significance is assessed by ordinary one-way ANOVA with Tukey's multiple comparisons test. (B) General intracellular ROS was assessed by flow cytometry using carboxy-H₂DCFDA probes in non-treated cells (NT), cells treated with 2.5 or 10 mM AA or DHA for 4 h (upper panel), cells treated with 10 mM AA, 10 mM AA plus 2000 U/ml catalase or 10 mM AA plus 10 mM GSH (lower panel). Treatment with 100 μM  H₂O₂ for 20 min was used as a positive control (PC). Mean fluorescence value in non-treated MDA-MB-231 cells is set as 1 and relative fluorescence intensity is represented. Data are means ± SD of 4 independent experiments. Statistical significance is assessed by ordinary one-way ANOVA with Tukey's multiple comparisons test. (C) MTT assay was performed on cells treated with 10 mM AA or DHA alone or in the presence of 2000 U/ml catalase, 10 mM NAC or 10 mM GSH for 24 h. Data are means ± SD of at least 6 independent experiments. Statistical significance is assessed by two-way ANOVA with Tukey's multiple comparisons test. ****, P < 0.0001; **, P < 0.01; *, P < 0.05.