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Fig. 2 | BMC Cancer

Fig. 2

From: Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferation

Fig. 2

Gap 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)

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