Figure 4: Tricellulin silencing does not lead to apical domain displacement at tricellular junctions.
From: Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity
(a) Confocal microscopy analysis of tricellulin distribution on the apical (Apical, upper panels) or lateral (Lateral, lower panels) side in control (Caco2 shNT, left panels) and EpCAM-depleted (Caco2 shEpCAM, right panels) cells. xy and xz views are presented. Scale bars, 5 μm. (b) 3D-SIM microscopy analysis of tricellulin localization in control (Caco2 shNT) and EpCAM-deprived (Caco2 shEpCAM) cells. While tricellulin was focused at the most apical part of the tricellular junctions in control cells, it was instead concentrated at the bottom of aberrant tricellular junctional tubes in the absence of EpCAM (white arrows). Scale bars, 2 μm. (c,d) Confocal microscopy analysis of double immunostainings for E-cadherin (green) and tricellulin (red) (c), occludin (green) and tricellulin (red) (d), on the basal or apical sides in EpCAM-depleted (Caco2 shEpCAM) cells. xy and xz views are presented. Scale bars, 2 μm. (e) Western blot analysis of the level of tricellulin expression in control (Caco2 shNT) or EpCAM-deprived (Caco2 shEpCAM #1 and Caco2 shEpCAM #2) clones. α-tubulin was used as loading control. (f) Statistical analysis of tricellulin amounts relative to α-tubulin amounts in control (Caco2 shNT) or EpCAM-deprived (Caco2 shEpCAM #1 and Caco2 shEpCAM #2) clones. The analysis was performed based on three independent experiments. Percentage of tricellulin expression in control cells=100%, in Caco2 shEpCAM#1 cells=121.8±0.18%, and in Caco2 shEpCAM#2=96.8±0.20%. Three independent experiments were used, and statistical significance was determined one-way analysis of variance and Tukey’s multiple comparison tests. (g) Western blot detection of tricellulin (upper panel) or EpCAM (lower panel) after immunoprecipitation of EpCAM or tricellulin from control Caco2 cell extracts. (h–m) Confocal microscopy analysis of EpCAM, E-cadherin, occluding, Crb3, villin and actin distribution on the apical (Apical, upper panels) or lateral (Lateral, lower panels) side in control (Caco2 shNT, left panels) and tricellulin-depleted (Caco2 shTricellulin, right panels) cells. xy and xz views are presented. Scale bars, 5 μm. Nuclei were detected with Hoechst 33342 staining (blue).
