Fig. 5. Mutant Cx43-eYFP do not form functional hemichannels as assessed by propidium iodide uptake. C6 cells, C6R control cells and clonal C6R cell lines expressing Cx43-eYFP constructs were analyzed for hemichannel function by determining the level of propidium iodide uptake after opening hemichannels by incubation of cell monolayers in 0 Ca²⁺. Stable expression of each construct in every cell was determined by detecting eCFP or eYFP fluorescence (A,D). A representative experiment showing PI uptake for control C6R (eCFP) (B,C) and wild-type Cx43-eYFP (E,F) after incubation in HBSS (B,E) with Ca²⁺ or HBSS (C,F) without Ca²⁺ containing PI for 15 minutes. Cells were fixed and uptake was visualized on a Nikon upright fluorescent microscope using rhodamine filter settings using a 20x objective. Digital photographs were quantitated using ImageJ software by averaging randomly selected average cell intensities for 40 cells per coverslip. (G) Quantitation of C6, C6R, wild type and each mutant is represented for HBSS with Ca²⁺ (white) and HBSS without Ca²⁺ (hatched). Data are expressed as mean±s.e.m. of several experiments on at least two cell lines for each mutant. Scale bar, 100 µm.