Differences in TJ length.

<p><b>A)</b> Upper image show immunostaining of ZO-1 (green) and nuclei staining (blue). Lower image shows magnifications of representative cell borders. <b>B)</b> Ratio of persistent length to contour length of all tested cell monolayers. 16HBE14o^– shows a cell-cell contact enlargement by 40% while CFBE41o^– cells and its transfected clones exhibit only a tiny enlargement by 10%. <b>C)</b> TJ lengths per area in µm per µm². 16HBE14o^– cells show 80% longer TJ lengths per area than CFBE41o^– cells. Furthermore, there is no difference in TJ lengths per area between CFBE41o^– cells and its transfected clones. Results are presented as mean ± SD (n = 7–8, p<0.05).</p

Scheme of the continuous transepithelial resistance measurement device (cTER).

<p>The ThinCert culture plate contains eight filter inserts with cell monolayers. The upper plate (lid) has six titanium electrodes for each insert, four electrodes to inject the current and two to measures the voltage. Electrodes are arranged in a way that resulted in a fairly homogenous electrical field.</p

Detection of tight junction proteins.

<p>Presence of tight junction proteins known to be expressed in alveolar epithelial cells (claudin-3, -4, -5, -7) and their junctional localization in 16HBE14o^–, CFBE41o^– cells and its transfected clones was verified by Western blot (A) and confocal laser scanning microscopy (B). For densitometric evaluation of Western blots (C), all signals were normalized to β-actin. All values are expressed relative to the respective value detected in 16HBE14o^– cell layers. One-way Anova analysis revealed that claudin-3 expression differed (p<0.05) in 16HBE14o^– and CFBE41o^– clones, whereas claudin-4, -5 and -7 expression was not significantly different (n = 4). No claudin-18 expression was detected (not shown).</p

Fluorescein flux per TJ length.

<p><b>A)</b> Calculation of flux per TJ length revealed a significant lower value for 16HBE14o^– cells under resting conditions (red box) compared to CFBE41o^– cells (green box). Stimulation with cAMP caused a 3 fold increase for 16HBE14o^– cells (red hatched box) while CFBE41o^– cells do not respond to stimulation (green hatched box). <b>B)</b> CFBE-WT cells (blue) and CFBE-delF (brown) do not show statistically significant differences in fluorescein flux per TJ length neither under resting conditions nor upon stimulation with cAMP (hatched boxes). Data are presented as a box-plot showing raw data (circles), median (horizontal line) 25 and 75 percentile (box) and SD (whiskers (n = 7–13, p<0.05).</p

Expression of CFTR mRNA.

<p>The relative quantity of CFTR gene expression was calculated by the 2^−ΔΔCt method, using GAPDH as the internal reference. CFTR mRNA expression value of 16HBE14o^– cells was defined as 1 and expression of all other cells were normalized to this value. CFTR mRNA levels of the other cell lines were displayed as a fold change relative to16HBE14o^–. Results are presented as mean ± SEM (n = 9–12, p<0.001).</p

Changes in transepithelial electrical resistance (cTER) upon cAMP.

<p><b>A)</b> 16HBE14o^– (triangles) and CFBE41o^– (circles) monolayer were grown on ThinCert supports. After 8–11 days in culture they obtained 800 and 500 Ω*cm² resistance respectively. After 5 min 8cpt-cAMP was added, causing dramatic TER decrease in 16HBE14o^– cells (red triangles) and an increase of TER for CFBE41o^– (green circles). Addition of the same amount of medium to control cells (open circles and triangles) did not show any effect. <b>B)</b> CFBE-WT monolayer (blue triangles) and CFBE-delF monolayer (brown circles) were grown on ThinCert supports. After 8–11 days in culture both clones obtained 500–600 Ω*cm² resistance. Stimulation with 8cpt-cAMP caused a decrease of TER in CFBE-WT cells (blue triangles) and an increase for CFBE-delF (brown circles). Addition of the same amount of medium to control cells (open circles and triangles) did not show any effect. Results are presented as mean ± SD (n = 3–6, p<0.05).</p