A. Functional GJIC between U87 and HMEC. Donor U87, transfected or not (Empty) with miR-5096-mimic (30 nM) or miR-5096-inhibitor (30 nM), were loaded with calcein and labelled with DiL-C18. Calcein diffuses through gap junctions, while DiL-C18 does not. These cells were then plated on unlabeled HMEC monolayer (acceptor). After 5 h of co-culture, HMEC establishing GJIC with U87 become fluorescent by calcein diffusion. Carbenoxolone (100 μM) prevented it without affecting the cell-to-cell adhesion. Dotted areas are enlarged in the right inserts (representative of 3 experiments; Bar 80 μm). B. Histogram shows the cell number of HMEC receiving dye (calcein) per U87 (mean ±SD; **P<0.01 vs control, n = 3). Carb.: 100 μM carbenoxolone. C. Immunoblot analysis of Cx43 protein in HMEC (left) and U87 (right) whole cell lysates after 12 h of co-culture. P0, P1 and P2 denote the three major Cx43 migration bands. One representative of 3 independent experiments is shown (β-actin as loading control). D. Histogram shows changes in all band intensity related to the total Cx43 expression level in the whole cell lysates (mean ± SD; *P<0.05 vs empty; Mann-Whitney U test and Kruskal-Wallis test; n = 3).