3.4 Confocal imaging shows that combination treatment induces changes in Cx43 expression and intracellular distribution.
To better understand the mechanism of αCT1 enhanced cell killing, we repeated the treatment in A172, U87, and BT169 cell lines using a fluorescence conjugated αCT1 (FITC-αCT1) combined with TMZ. The interaction between FITC-αCT1 and N-terminal Cx43 was visualized under the confocal microscope. The complete procedure can be found in sections 2.6 and 2.7.
As shown in Figure 5 , Cx43 level did not show significant changes after the TMZ (100 μM), or FITC-αCT1 (100 μM) treatment in any of the three cell lines. However, in A172 and BT169 cell lines (Figure 5A and 5B ), the combination of 100 μM TMZ and 100 μM FITC-αCT1 treatment group significantly increased the number of Cx43 aggregates as well as the Cx43 signal intensity compared to the U87 cell line, which only exhibits a slight increase in the Cx43 aggregates number within the cell bodies (Figure 5C ). This observation indicates that FITC-αCT1 combined with TMZ may induce changes in TMZ- related signaling pathways and affect Cx43 protein functions and activities in GBM cells.