Effects of claudin-2 and claudin-4 double knockout on the barrier properties of TJs.

<p>(A) Time course of TER and TER values at 6 days and 14 days after the seeding on filter inserts in claudin-2 knockout clones (CTL1 and CTL2) and claudin-2 and claudin-4 double knockout clones (dKO1–3). (B and C) <i>P</i>_Na/<i>P</i>_Cl, <i>P</i>_Na and <i>P</i>_Cl at 6 days (B) and 14 days (C) after the seeding on filter inserts in claudin-2 knockout clones and claudin-2 and claudin-4 double knockout clones. (D) Flux of 4 kDa FITC-dextran in claudin-2 knockout clone and claudin-2 and claudin-4 double knockout clones. N = 3–4 for each experiment.</p

Claudin-4 knockout by TALEN-mediated gene targeting in MDCK cells: Claudin-4 is dispensable for the permeability properties of tight junctions in wild-type MDCK cells

<div><p>Epithelia act as a barrier between the internal and external environments, and the movement of substances via the paracellular pathway is regulated by tight junctions (TJs). Claudins are major determinants of TJ permeability. Claudin-4 was the first claudin whose involvement in the TJ permeability in cultured cells was directly demonstrated, but the permeability properties of individual claudins including claudin-4 are still incompletely clarified. In this study, we established claudin-4 knockout cells using transcription activator-like effector nucleases (TALENs), a recently developed method for genome editing, and investigated the permeability property of claudin-4 in MDCK II cells. We found that claudin-4 knockout has no apparent effect on the localization of other claudins and electrophysiological properties in MDCK II cells. Therefore we further established claudin-2 and claudin-4 double knockout clones and investigated the effects on TJs. Claudin-4 knockout in addition to claudin-2 knockout slightly increased the localization of other claudins at TJs but showed no obvious effects on the electrophysiological properties in MDCK II cells. These results indicate that claudin-4 is dispensable for the barrier property of TJs in wild-type as well as claudin-2 knockout MDCK II cells. Our results suggest the need for further knockout analysis to reveal the permeability properties of individual claudins.</p></div

Establishment of claudin-2 and claudin-4 double knockout clones in MDCK II cells.

<p>(A) Immunofluorescence analysis of claudin-4 and occludin in claudin-2 knockout clone (CTL) and claudin-2 and claudin-4 double knockout clones (dKO1–3) in MDCK II cells. Claudin-4 staining at cell-cell contacts was completely lost in double knockout clones. Scale bar = 10 μm. (B) Immunoblots of claudin-4 and E-cadherin in claudin-2 knockout clone and double knockout clones. (C) DNA sequences of the TALEN targeting site in wild-type cells and double knockout clones. One type of mutation was found in the alleles of the dKO3 clone and two types in the alleles of the dKO1 and dKO2 clones. Green letters indicate additional nucleotides. Frame shifts were confirmed in all alleles. (D) Genomic PCR analysis of wild-type cells and double knockout clones using primers for TALEN and claudin-4 DNAs. None of the PCR products for TALENs was detected in double knockout clones.</p

Establishment of claudin-2 and claudin-4 double knockout clones in MDCK II cells.

<p>(A) Genomic PCR analysis of wild-type cells, claudin-4 knockout clones (sKO4, sKO5) and claudin-2 and claudin-4 double knockout clones (dKO4–6) using primers for the TALEN targeting site in claudin-4 genes. (B) DNA sequences of the TALEN targeting site in wild-type cells and dKO4 and dKO6 clones. Green letters indicate additional nucleotides. (C) Genomic PCR analysis of wild-type cells and sKO4, sKO5 and dKO5 clones using primers for the region of sequences containing 400 bases before the TALEN targeting site. (D) Genomic PCR analysis of wild-type cells and sKO4, sKO5 and dKO5 clones using primers for the region within the claudin-4 gene. (E) Immunoblots of claudin-1, -2, -3, -4 and -7 in claudin-4 knockout clones.</p

Effects of claudin-4 knockout on the localization of other TJ proteins and cytoskeleton.

<p>(A) Immunofluorescence analysis of claudin-1, -2, -3, -7, ZO-1, occludin, F-actin, and myosin heavy chain II-B (MHC-B) in wild-type cells (CTL) and claudin-4 knockout clone 1 (sKO1). The localization of these proteins was not apparently changed in the claudin-4 knockout clone. Scale bar = 10 μm. (B) Immunoblots of claudin-1, -2, -3, and -7 in wild-type cells and claudin-4 knockout clones.</p

Effects of claudin-2 and claudin-4 double knockout on the localization of other claudins.

<p>(A) Immunofluorescence analysis of claudins in the co-culture of the claudin-2 knockout clone 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182521#pone.0182521.ref013" target="_blank">13</a>] and the dKO1 clone (claudin-2 and claudin-4 double knockout clone). Scale bar = 10 μm. (B) Quantification analysis of the signal intensity of claudins at TJs in the claudin-2 knockout clone 1 (CTL1) and the dKO1 clone. N = 4 for each experiment.</p

Effects of claudin-4 knockout on the localization of other claudins.

<p>(A) Immunofluorescence analysis of claudins in the co-culture of wild-type MDCK II cells and claudin-4 knockout clone 3 (sKO3). No apparent difference was observed between the localization of claudin-1, -2, -3 and -7 in the wild-type cells and claudin-4 knockout clone. Scale bar = 10 μm. (B) Quantification analysis of the signal intensity of claudins at TJs in wild-type cells and claudin-4 knockout clone. The signal intensity of claudins at TJs in wild-type cells and claudin-4 knockout clone 3 was measured as described in <i>Materials and Methods</i>, and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in wild-type cells (CTL) and claudin-4 knockout clone 3 (sKO3) to the signal intensity in wild-type cells. The signal intensity of claudins at TJs was not significantly different between wild-type cells and claudin-4 knockout clone. N = 4 for each experiment.</p

Establishment of claudin-4 knockout clones in MDCK II cells.

<p>(A) Immunofluorescence analysis of claudin-4 and occludin in wild-type cells (CTL) and claudin-4 knockout clones (sKO1–3) in MDCK II cells. Claudin-4 staining at cell-cell contacts was completely lost in claudin-4 knockout clones. Scale bar = 10 μm. (B) Immunoblots of claudin-4 and E-cadherin in wild-type cells and claudin-2 knockout clones. A claudin-4 band of ~20 kDa was absent in claudin-4 knockout clones. (C) DNA sequences of the TALEN targeting site in wild-type cells and claudin-4 knockout clones. Dash indicates loss of a nucleotide and green letters indicate additional nucleotides. Frame shifts were confirmed in all clones. (D) Genomic PCR analysis of wild-type cells and claudin-4 knockout clones using primers for TALEN and claudin-4 DNAs. A clone stably expressing TALEN was used as a positive control (PC). None of the PCR products for TALENs was detected in claudin-4 knockout clones.</p

Effects of claudin-2 and claudin-4 double knockout on the localization of other TJ proteins and cytoskeleton.

<p>(A) Immunofluorescence analysis of claudin-1, -2, -3, -7, ZO-1, occludin, F-actin, and myosin heavy chain II-B (MHC-B) in claudin-2 knockout clone (claudin-2 KO) and claudin-2 and claudin-4 double knockout clone 1 (dKO1). The localization of claudin-7 in cell-cell contacts at the TJ level showed clearer sharp lines in the double knockout clone than the claudin-2 knockout clone. Scale bar = 10 μm. (B) Immunoblots of claudin-1, -3, and -7 in claudin-2 knockout clone and double knockout clones. The expression levels of claudin-1, -3 and -7 were increased in the dKO1 clone whereas those in the dKO2 and dKO3 clones were not changed compared with the claudin-2 knockout clone.</p

Time course and reversibility of epithelial stratification by the hydrostatic pressure.

<p>(A) A vertical section of MDCK I cell sheets. Hydrostatic pressure from basal to apical side was applied to the MDCK I cell sheets at two days after seeding on filters, and the vertical section of cell sheets was observed at Day 2 and 1–12 days after application of the hydrostatic pressure (Days 3–14). MDCK I cells showed gradual development of epithelial stratification with time. (B) The reversibility of epithelial stratification by the hydrostatic pressure. Hydrostatic pressure from basal to apical side was applied to the MDCK I cell sheets for four days, and the hydrostatic pressure gradient was then eliminated by the increase of the culture medium in the apical side. (C) Stratification index under the conditions in (A) and (B). The upper side is apical side and the lower side is basal side. Scale bars = 20 μm.</p