Time-dependent and rapid decrease in Cx43 protein levels in ethanol-treated HL-1 cells.

<p>(A-D) Cells were incubated in medium containing 2% ethanol for the indicated time periods, and the protein levels of Cx43, Cx40 and Cx45 were determined by western blot analysis (A). Hsc70 served as a loading control. Quantitative analysis of Cx43 (B), Cx40 (C) and Cx45 (D) is shown. Each graph shows mean±S.E. (n = 4–6). **, p<0.01 versus 0 h. Relative levels to control (0 h) group were shown for Cx43 (B). (E) The levels of Cx43 mRNA were determined by qPCR. GAPDH served as an internal control. The graph shows mean±S.E. (n = 3). **, p<0.01, *, p<0.05 versus 0 h. (F) Ethanol disrupts cell-cell coupling. Cells were incubated in the medium containing 2% ethanol for 48 hours, and scrape-loading dye transfer assay was performed as described in the Materials and methods. Areas of dye spread (fluorescent areas) were quantified using ImageJ software. The graph shows mean±S.E. (n = 5). **, p<0.01 versus control.