Down-regulation of claudin-2, ZO-3, and claudin-3 in cingulin/paracingulin double knockdown MDCK cells.

<p>(<b>A</b>) Histogram showing relative mRNA levels, determined by qRT-PCR, for the indicated transcripts, in wild-type MDCK cells (WT), in a MDCK cell clone expressing a control shRNA (Control), in a cingulin single-KD cell clone (CGN(-)), in a paracingulin single-KD cell clone (CGNL1(-)), and in a double-KD cell clone (CGN(-)/CGNL1(-)). The relative mRNA levels were calculated as the ratio of the mRNA in experimental samples versus WT cells (taken as 100%). *p<0.05, compared with mRNA of WT cells. (<b>B</b>) Immunoblotting analysis of TJ proteins in WT cells, in a clone expressing a control shRNA (Control), in two independent (a and b) paracingulin single-KD cell clones (CGNL1(-)), and in two independent (A and B) double-KD cell clones (CGN(-)/CGNL1(-)). (<b>C</b>) Immunofluorescence localization of CGNL1, CGN, claudin-2, ZO-3, ZO-1 and occludin in WT, control, CGN(-), CGNL1(-) and CGN(-)/CGNL1(-) cells. (<b>D</b>) Immunoblotting analysis and (<b>E</b>) immunofluorescence localization of α-catenin (α-cat), β-catenin (β-cat), and E-cadherin (E-cad) in wild-type (WT) MDCK cells, in a MDCK cell clone expressing a control shRNA (Control), and in a double-KD (CGN(-)/CGNL1(-)) cell clone. Bar (C) and (E) = 10 µm. Protein loadings in (B) and (D) were normalized by immunoblotting samples with anti-β-tubulin antibodies (shown in (B) only). The behaviour of additional double-KD clonal lines was very similar to the representative one shown here. (<b>F</b>) qRT-PCR and (<b>G</b>) immunoblotting analysis of WT, paracingulin single-KD (CGNL1(-)) and cingulin/paracingulin double-KD (CGN(-)/CGNL1(-)) cells with or without additional expression of the tetracycline repressor (TetR), which blocks the expression of shRNA. The relative mRNA levels were calculated as the ratio of the mRNA in experimental samples to that in WT cells (taken as 100%). *p<0.05, compared with WT cells. Protein loadings in (G) were normalized by immunoblotting with anti-β-tubulin antibodies.