*4.3. Immunofluorescent Staining*

Immunofluorescence analysis was carried out as described before [21]. Immunofluorescence studies were performed on culture-plate inserts. Confluent monolayers were rinsed with PBS, fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized for 10 min with PBS containing 0.5% (*v*/*v*) Triton X-100. To block non-specific binding sites, cells were then incubated in PBS containing 1% ( *w*/*v*) BSA and 5% (*v*/*v*) goa<sup>t</sup> serum (blocking solution; Biochrom) for 60 min. All subsequent washing procedures were performed with this blocking solution.

After blocking, cells were incubated overnight at 4 ◦C with primary antibodies for angulin-1 (1:1000; Sigma-Aldrich), tricellulin (1:500; Thermo Fisher Scientific, Invitrogen), occludin (1:250; Thermo Fisher Scientific, Invitrogen) and ZO-1 (1:250; Thermo Fisher Scientific, Invitrogen), followed by washing steps and incubation during 60 min at room temperature with the respective secondary antibodies (Alexa Fluor 488 goa<sup>t</sup> anti-rabbit and Alexa Fluor 594 goa<sup>t</sup> anti-mouse, each 1:500; Thermo Fisher Scientific, Waltham MA, USA) and <sup>4</sup>,6-diamidino-2-phenylindole (DAPI, 1:1000; Roche, Mannheim, Germany). Cell culture inserts were mounted on microscope slides using ProTag MountFluor (Biocyc, Luckenwalde, Germany). Images were obtained with a confocal laser-scanning microscope (LSM 780, Zeiss, Jena, Germany) and processed using ZEN software (Carl Zeiss, Oberkochen, Germany, ZEN black edition 2012 SP1, ver. 8.1).

#### *4.4. Freeze Fracture Electron Microscopy*

At a confluency of 100%, cells were washed with PBS (containing Ca2+/Mg2+) and fixed with 2.5% glutaraldehyde at RT for 2 h. After washing twice with PBS (containing Ca2+/Mg2+), the cells were stored at 4 ◦C in 0.25% glutaraldehyde. Small rectangles of the bottom of the cell culture filters were cut out, the attached cells were cryoprotected in 30% glycerol for 30 min, placed between two gold specimen holders and shock frozen in R422D (TEGA GmbH, Würzburg, Germany) cooled by liquid nitrogen ( −210 ◦C).

The samples were fractured using the freeze-fracture device Denton DV-502 (Denton Vacuum, Moorestown, NJ, USA) at −100 ◦C and 2 × 10−<sup>10</sup> Torr. The samples were vaporized at −150 ◦C (2 × 10−<sup>10</sup> Torr) with a layer of platinum and then a layer of carbon. This results in a thin metal film on the broken sample. The replicas were cleaned with 12% sodium hypochlorite, washed several times in ddH2O and mounted on a copper mesh grid. The Zeiss 902A electron microscope was used to examine the replicas at 80 kV. Magnifications between 20,000 and 50,000 was used.

#### *4.5. Measurement of 4-kDa FITC-Dextran Flux*

Flux studies were performed in conventional Ussing chambers for cell-culture inserts [45] under voltage clamp conditions. Dextran flux was measured in 5 mL circulating 111-Ringer's ((in mM) 119.7 NaCl, 21.4 NaHCO3, 2.5 Na2HPO4, 0.6 NaH2PO4, 5.7 KCl, 1.3 MgCl2, 1.2 CaCl2, and 10.0 D(+)-glucose) containing also 37 mM unlabeled 4-kDa dextran (SERVA, Heidelberg, Germany) on both sides of the cells for isosmotic conditions and only on the apical side for osmotic conditions. After addition of 0.2 mM 4-kDa FITC-labeled dialyzed dextran (Sigma-Aldrich) to the apical chamber (final concentration), basolateral samples (200 μL) were collected at 0-, 20-, 40-, 60-, 80-, 100-, 120- and 140-min. Tracer fluxes were determined from FITC-dextran samples, which were measured with a fluorometer at 520 nm (Spectramax Gemini, Molecular Devices, Ismaning, Germany). Dextran permeability was calculated from *p* = J/Δc with *p* = permeability (cm·s<sup>−</sup>1), J = flux (mol·h−1·cm<sup>−</sup>2) and c = concentration (mol/L).

#### *4.6. Dilution Potential Measurements*

Dilution potential measurements for the determination of ion permeabilities were performed in Ussing chambers for cell-culture inserts [45]. Water-jacketed gas lifts kept at 37 ◦C were filled with 10 mL circulating fluid on each side. The bathing solution contained (in mM) 119.7 NaCl, 21.4 NaHCO3, 5.7 KCl, 1.3 MgCl2, 1.2 CaCl2, 3.0 HEPES, and 10.0 D(+)-glucose, and was gassed with 95% O2 and 5% CO2 to ensure a pH value of 7.4. All experimental data were corrected for the resistance of the empty filter and the bathing solution. Dilution potentials were measured with modified bathing solution on the apical or basolateral side of the epithelial monolayer. In the modified bathing solution, NaCl was iso-osmotically replaced by mannitol. The ratio of PNa and PCl and the absolute permeabilities for Na+ and Cl− were calculated as described before [46].

#### *4.7. Measurement of Transepithelial Water Transport*

Water flux measurements were performed using a modified Ussing chamber developed by our group [12–14,21]. The gas lifts of the common Ussing chamber were replaced by two glass tubules with a small diameter, where the menisci of the perfusion solution are clearly visible. From changes in the menisci, the water flux is calculated. Throughout these experiments, transepithelial voltage (mV) was clamped to 0 mV and transepithelial resistance (TER, <sup>Ω</sup>·cm2) and short-circuit current (ISC, <sup>μ</sup>A·cm<sup>−</sup>2) were recorded. Resistances of bathing solution and blank filter support were measured prior to each experiment and subtracted. The stability of TER served as an indicator of cell viability.

Cell filters were mounted in Ussing chambers and perfused with HEPES-buffered solution with the following composition (in mM): 144.8 NaCl, 2.4 Na2HPO4, 0.6 NaH2PO4, 5.4 KCl, 1.2 MgCl2, 1.2 CaCl2, 10.6 HEPES, and 10.0 D(+)-glucose. The pH value of the perfusion solution was pH 7.4. A rotary pump ensured constant circulation of the perfusion solution (4.0 mL·min−1) and thus a fast fluid exchange in both hemichambers (volume 500 μL) to avoid effects of unstirred layers on water permeability. Water flux was induced by a transepithelial osmotic gradient: (i) 100 mM mannitol, (ii), 37 mM 4 kDa-dextran or (iii) 100 mM 4 kDa-dextran. The solution was added in the apical or basolateral compartment of the Ussing chamber. The osmolality of the perfusion solutions (mosmol/kg, abbreviated mOsm) was determined using a freezing point depression osmometer (Osmomat 3000, Gonotec, Berlin, Germany). The osmolality of the HEPES-buffered solutions is annotated in Table 3.


**Table 3.** Osmolality of the HEPES-buffered solutions using a freezing point depression osmometer.

The fluid level in both glass tubes was monitored by a visual system ColorView XS (Olympus Soft Imaging Solutions GmbH, Munster, Germany) at time 0 min and with intervals of 10 min over a period of 120 min. Transepithelial water flux, given as flux per square centimeter and hour, was calculated after special calibration from the difference between the menisci at the registration times. Fluxes directed from the basolateral to the apical compartment were defined as positive flux.
