Reduced accumulation of the components of the nectin and cadherin systems at the dendritic filopodia-filopodia contact sites in ZO-1-knockdown neurons.

<p>Cultured ZO-1-knockdown hippocampal neurons on 7 DIV were prepared as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076201#pone-0076201-g004" target="_blank">Figure <b>4A</b></a> and double-stained for EGFP and one of nectin-1, nectin-3, afadin, N-cadherin, β-catenin or αN-catenin. (A1) EGFP and nectin-1; (A2) EGFP and nectin-3; (A3) EGFP and afadin; (B1) EGFP and N-cadherin; (B2) EGFP and β-catenin; (B3) EGFP and αN-catenin; (<b>a</b>) low magnification images; (<b>b</b>) high magnification images of the boxed areas in (<b>a</b>). <b>Upper </b><b>rows</b>, control neurons; <b>lower </b><b>rows</b>, ZO-1-knockdown neurons. <b>Bars</b>, <b>upper </b><b>rows</b> 10 µm; <b>lower </b><b>rows</b> 2.5 µm. Arrowheads indicate dendritic filopodia-filopodia contact sites. (<b>C</b>) Quantitation of the co-localization of the components of the nectin and cadherin systems with ZO-1 in ZO-1-knockdown neurons. The data are presented as mean plus SEM (error bars) for each sample (n = 30 for nectin-1, afadin, β-catenin and αN-catenin; n = 40 for nectin-3; n = 20 for N-cadherin) *, p value < 0.05; and **, p value < 0.01 (Student’s t-test) (AU) arbitrary unit. It is noted that the immunofluorescence signals for the components of the nectin and cadherin systems were still observed at some dendritic filopodia-filopodia contact sites in the ZO-1 knockdown neuronal cultures as shown in (<b>A</b>) <b>and </b>(<b>B</b>). We estimated that the transfection efficiency of the siRNA in cultured neurons was about 80%. Thus, ZO-1 might not be knocked-down in some neurons, in which these immunofluorescence signals were observed.