Dye transfer assays for gap junction forming mutants.

<p>Intercellular communication was assayed by scrape dye loading using Lucifer Yellow (<b>LY</b>) and Dextran Texas Red (<b>TxR</b>). LY will pass through gap junctions while Dextran TxR will not. (<b>Panel A</b>) Confluent gap-junction deficient parental HeLa cells (left column) and HeLa cells transiently transfected with Cx26 WT-GFP-4C (right column) serve as negative and positive controls, respectively. Top row: Images of LY fluorescence. Middle row: Images of TxR fluorescence Bottom row: Differential Interference Contrast (DIC) light micrograph of the same field of cells showing cell-cell contact. Dextran Texas Red acts as a reporter for dead or non-communicating cells and those along the scratch that also contained Lucifer Yellow were not counted in this analysis. (<b>Panel B</b>) Similar images as in (Panel A) for the two mutants, M195T and A197S, which made detergent stable hemichannels and channels. (<b>Panel C</b>) LY (top), TxR (middle) and DIC images (bottom) for mutants that made detergent unstable hemichannels and channels (left to right, C202F, I203T, L205V and N206S). The inset at the left is a 4.5x magnification of a LY non-transferring cell expressing C202F gap junctions. The arrow points to these cells in the C202F LY image. (<b>D</b>) Histogram of levels of communicating cells for each mutant. Error bars are standard errors of the mean (SEM).