Fig. 8. Compromise of TJ integrity following knockdown and rescue of Bves in HCE cells. (A) Expression of Bves and ZO-1 at the cell periphery in parental HCE cells. (B) HCE-R cells stably expressing the `rescue' chicken Bves-FLAG construct exhibit labeling of the membrane by both FLAG and Bves antibodies. Note the intracellular accumulation of transfected chicken Bves. (C) Treatment with anti-human Bves MO results in a loss of Bves at the cell periphery, as compared to untreated (NT) and control MO. (D) TER measurements were performed on HCE and HCE-R. Parental HCE cells exhibited a TER value of approximately 390 /cm². Treatment with anti-human Bves morpholino resulted in a 40% decrease (P<0.05) in TER (asterisk), while control MO did not significantly alter the TER. The TER of HCE-R cells doubled to 775 /cm² and, following anti-human Bves MO (double asterisk), a drop of only 20% (P<0.05) in TER was observed. (E) ZO-1 immunofluorescence performed on Transwell inserts following TER measurements. Parental HCE cells and HCE-R cells were treated with control and anti-human Bves MO. Bves and ZO-1 expression is disrupted in HCE cells treated with anti-human Bves MO (top right panel). Morpholino treatment does not alter the expression pattern of ZO-1 in HCE-R cells (lower right panel). (F) Western blotting demonstrates that membrane ZO-1 and Bves increase with exogenous expression of Bves in HCE-R cells, but decrease following treatment with Bves-MO, as compared with the parental HCE line. (G) Membrane integrity of junction proteins occludin, E-cadherin and ß-catenin is disrupted following Bves MO treatment. Scale bars: 10 µm.