Figure 7

Dye transfer between 4T1-GFP and pulmonary microvascular endothelial cells. (A) Subconfluent monolayers of pulmonary microvascular endothelial cells in coverglass-bottomed dishes was labeled with 5 μM Calcein Red AM and then thoroughly washed for removal of extracellular dye. 4T1-GFP cells with or without connexin-43 (Cx43) mutation variants were deposited into the dish and placed in a cell culture incubator. After 1 hour, the cells were imaged in the red (for Calcein Red) and green fluorescent protein (GFP) channels and the pictures were overlaid. In tumor cells (green) that were in close contact with endothelial cells, transfer of Calcein Red can be observed (red color inside green cells). (B) Quantification of 4T1-GFP cells exhibiting dye transfer from endothelial cells. Dye transfer was significantly reduced in 4T1-GFP tumor cells expressing the G138R Cx43 mutant (red bar) and in the presence of gap junctional inhibitor 18β-glycyrrhetinic acid (30 μM) (blue bar). On the other hand overexpressing wild-type Cx43 enhanced dye transfer (green bar). The expression of C61S mutant did not seem to affect dye transfer (yellow bar).