Exogenous connexin43 (Cx43) in HBL100 cells forms gap junctional plaques. To explore if Cx43 expression and/or gap junctional intercellular communication (GJIC) affects diapedesis of breast tumor cells we used GJIC-deficient HBL100 cells to engineer cells expressing functional Cx43 (HBL100Cx43) and non-functional Cx43 (HBL100GFP-Cx43). HBL100v, containing the empty expression vector, served as a control. (a) Cx43 expression of these cells was compared by western blot analysis to that of human microvascular endothelial cell (HMVEC) grown on matrigel used as a control for endogenous Cx43 expression. Whereas Cx43 was undetectable in HBL100 and HBL100v cells, HMVEC grown on matrigel, HBL100Cx43, and HBL100GFP-Cx43 cells expressed Cx43. Immunocytochemistry localized Cx43 at cell-cell borders in (b) HMVEC monolayers (arrows), (e) between HBL100Cx43 cells (arrow) or (f) HBL100GFP-Cx43 cells (arrows), whereas (c) HBL100 and (d) HBL100v cells lacked Cx43 staining. Bar = 10 μm.