Fig. 7
Activation of Cx43-formed hemichannels in endothelial cells of migration front depends on NO-mediated S-nitrosylation. a, Representative images of ethidium uptake in primary cultures of mesenteric endothelial cells (left) and the analysis of ethidium uptake rate observed in the migration front and in the monolayer in control conditions and after the treatment with 100 µM NG-nitro-L-arginine (L-NA) or 50 µM ascorbic acid (AA). The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity along the time. b, Representative images (left) and fluorescence intensity analysis (right) of the increase in [Ca2+]i observed in endothelial cells of the migration front in control conditions and in the presence of 100 µM L-NA or 50 µM AA. Variations in the levels of [Ca2+]i were assessed with the fluorescent Ca2+ indicator Fluo-4. c, Representative images (left) and quantitative analysis (right) of the endothelial cell migration observed in the wound-healing assay just after scratching the monolayer (0 h) and after 15 h in control conditions and in the presence of L-NA or AA. Yellow lines are only intended to highlight the migration front and are not a reference for migration analysis. d, Detection of total protein S-nitrosylation by immunofluorescence (left) and densitometric analysis of the fluorescence intensity (right) observed in the migration front and the monolayer of primary cultures of endothelial cells in control conditions and after the treatment with L-NA or AA. Cell nuclei are highlighted by the staining with DAPI (blue). Numbers inside the bars indicate the n value. Values are means ± SEM. *, P < 0.05 and **, P < 0.01 vs. Control by one-way ANOVA plus Bonferroni post hoc test