Fig. 2From: Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferationGap junction communication between mural cells and endothelial cells requires mural cell Cx43 and is decreased by tumor cells. Untransfected vSMC or vSMC nucleofected with non-targeting (‘siNT’) or Connexin43-specific (‘siCx43’) siRNA were treated with control (‘Mock’) or media conditioned by MDA-MB-231 cells (‘231 CM’), loaded with calcein-AM, and plated onto PKH26-labeled HUVEC. Dye transfer from vSMC to HUVEC was quantified using flow cytometry. a. Representative flow cytometry dot plots showing dye transfer as percentage of double-labeled cells in upper right quadrant. b. Ratio of dye transfer as calculated in Materials and Methods. Data represent the mean of four to five independent experiments performed in triplicate +/−s.e.m. (duplicate for 18-GA); ***p < 0.001. c. Western analysis of Connexin 43 in vSMC samples incubated in parallel with experiment from A. d. vSMC and GFP-HUVEC were plated in co-culture for 72 h as in ‘Methods’, then stimulated for 24 h with control media (‘Mock’) or media conditioned by MDA-MB-231 cells (‘231 CM’), and the number of heterocellular Cx43-expressing junctions (red) extending between endothelial cells (green) and mural cells quantified by confocal Z-stack analysis. Top panel, representative confocal images; inset, magnification of z-stack image of Cx43 gap junction. Bottom panel, quantification of Cx43-positive heterotypic gap junctions as seen in top panel. Data represent the quantification from paired cells over three experiments (n = 46–48 cell pairs each condition)+/−s.e.m. (*p < 0.05)Back to article page