Fig. 3

Cx43-formed hemichannels, but not gap junction channels, are associated with endothelial cell migration. a, Representative images of dye coupling assay (left) and the analysis of the number of coupled cells via gap junction communication (right) attained in the intact monolayer and in the migration front after scratching the monolayer. Dye coupling was assessed by measuring after 2 min the diffusion to neighboring cells (coupled cells) of the ethidium bromide microinjected into a single endothelial cell. The yellow diamond indicates the microinjected cell. b, Representative images of the ethidium uptake observed in primary cultures of mesenteric endothelial cells in control conditions and in the presence of the Cx blocking peptide ^37,43Gap27 (200 µM) or the Cx43 hemichannel inhibitor TAT-Gap19 (Gap19, 300 µM) (left). Ethidium uptake was evaluated 15 min after scratching the monolayer and cells were incubated with the dye for 15 min, as shown in the time course of ethidium uptake observed in the intact monolayer and in the migration front (b, right top). Dot yellow lines depict the edge of the migration front. In addition, the analysis of the ethidium uptake rate achieved in the intact monolayer and in the migration front in control conditions and in the presence of ^37,43Gap27 or Gap19 is also shown (b, right bottom). The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity along the time. Changes in ethidium-fluorescence signal are expressed in arbitrary units (a.u.). Numbers inside the bars indicate the n value. Values are means ± SEM. ***, P < 0.001 vs. Monolayer by two-way ANOVA. ††, P < 0.01 and †††, P < 0.001 vs. Migration front in control conditions (Control) by one-way ANOVA plus Bonferroni post hoc test. &, P < 0.001 vs. Migration front by paired Student’s t-test