Figure 6

Expression of connexin-43 by the mutant variants. (A) Immunocytofluorescence analysis of connexin-43 (Cx43) expression in wild-type 4T1-GFP tumor cells and in 4T1-GFP cells transfected with expression vectors of the mutant G138R and C61S in addition to a vector expressing two copies of wild-type Cx43 (Cx43OE). Endogenous Cx43 in wild-type 4T1-GFP cells is present mostly at the plasma membrane between the cells (3A, 4T1, white arrows). The G138R dominant-negative mutant was expressed both at the plasma membrane (white arrows) and in the cytoplasm (yellow arrow). Wild-type overexpression led to upregulation of Cx43 in both the plasma membrane (white arrows) and in the cytoplasm (yellow arrows, 3A, Cx43OE). Cx43 with the C61S mutation was mostly in the cytoplasm (yellow arrows, 3A, C61S). (B) Quantification of the immunocytofluorescence signal of Cx43 in 4T1-GFP cells expressing mutant and wild-type Cx43. All of the transfectants showed at least a factor or two more Cx43 expression compared with endogenous expression (control) or with mock transfection. (C) Western blot analysis showing increased expression of Cx43 forms in transfected cells when compared with non-transfected 4T1-GFP cells. The quantification of bands from three blots illustrates that the transfected cells show an at least two-fold increase in Cx43 expression compared with wild-type 4T1-GFP cells. *P < 0.05.