For immunofluorescence (IF) analysis, MDCK clonal lines were co-cultured with WT cells on 12 mm Transwell filters seeded at a density of 1 × 105 cells/well. For rescue IF experiments, double-KO cells were seeded and transfected 24 h later with 0.2 µg of plasmid DNA using jetOPTIMUS (Polyplus, VWR International AG, Nyon, Switzerland, #117-15). At confluence (72 h post-seeding and 48 h post-transfection), cells were washed with icecold PBS and fixed using cold methanol overnight at −20 ◦C, followed by a quick incubation in ice-cold acetone (1 min). Filters were excised manually using a razor blade, followed by rehydration in IMF buffer (0.1% TX-100, 0.15 M NaCl, 5 mM EDTA, 20 mM Hepes, pH = 7.5) and 2 washes in IMF buffer. Incubations with primary antibodies, anti-claudin-2 (Mouse, #32-5600, 1/50), anti-CGN (Rabbit, #C532, 1/5000), anti-GFP (Rabbit, #A11122, 1/200), anti-Myc (Rabbit, #06-549, 1/100), and anti-PLEKHA6 (Rat, #RtSZR127, 1/100) [32], were carried out at room temperature for 3 h (in a humidified chamber), followed by 3 washes in IMF buffer. Then, the filters were incubated with DAPI and secondary antibodies (1/300) for 2 h at room temperature, followed by 4 washes in IMF buffer.

Cingulin (CGN) and paracingulin (CGNL1) are cytoplasmic proteins of tight junctions (TJs), where they play a role in tethering ZO-1 to the actomyosin and microtubule cytoskeletons. The role of CGN and CGNL1 in the barrier function of epithelia is not completely understood. Here, we analyzed the effect of the knock out (KO) of either CGN or CGNL1 or both on the paracellular permeability of monolayers of kidney epithelial (MDCK) cells. KO cells displayed a modest but significant increase in the transepithelial resistance (TER) of monolayers both in the steady state and during junction assembly by the calcium switch, whereas the permeability of the monolayers to 3 kDa dextran was not affected. The permeability to sodium was slightly but significantly decreased in KO cells. This phenotype correlated with slightly increased mRNA levels of claudin-2, slightly decreased protein levels of claudin-2, and reduced junctional accumulation of claudin-2, which was rescued by CGN or CGNL1 but not by ZO-1 overexpression. These results confirm previous observations indicating that CGN and CGNL1 are dispensable for the barrier function of epithelia and suggest that the increase in the TER in clonal lines of MDCK cells KO for CGN, CGNL1, or both is due to reduced protein expression and junctional accumulation of the sodium pore-forming claudin, claudin-2.