Fig. 2

Inhibition of Cx43 expression in breast cancer cells inhibits GJ communication in vitro and inhibits brain colonization in mice. (A). 4T-1 cells were either treated with an empty lentiviral vector (Control) or treated with the lentiviral vector encoding shRNA to Cx43 (4T-1KNcx43) to knock down Cx43 expression. Stable cells lines were then selected and Cx43 expression levels examined by western blotting. Actin, GAPDH and Cx26 served as specificity and loading controls. 4T-1KNcx43 cells show a 78% decrease in Cx43 expression compared with 4T-1 control cells, as measured by densitometry. (B) The indicated tumor cells were prelabeled with calcein orange dye and then added to a monolayer of EA.hy926 endothelial cells in the presence of the GJ inhibitor CBX (10μM) or vehicle PBS. Dye transfer from tumor cells to endothelial cells was observed live by epifluorescence microscopy after 30minutes of co-culture. The number of adherent cells that transferred dye to the adjacent endothelium was determined and represented as percentage of total number of tumor cells counted. (C) The indicated tumor cells were cultured in vitro and examined for cell growth for 3days in the presence of CBX (10μM) or vehicle using the CyQUANT assay. rfu, relative florescence units. (D) Average number of micrometastatic lesions in the mouse brain induced by 4T-1 and 4T-1KNcx43 cells at 3–7 days post injection. Data indicate means + s.e.m. *P<0.05.