Journal: International Journal of Molecular Sciences

Article Title: Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells

doi: 10.3390/ijms21020527

Figure Lengend Snippet: Temporal redistribution of TJ proteins and barrier dysfunction caused by EPEC. ( A–C ) SKCO-15 cells were plated on Transwells and infected or not (UI) with EPEC. Total JAM-A, JAM-A S285, JAM-A Y280, occludin, and ZO-1 localization and TER were determined. ( A ) EPEC does not alter the distribution of total JAM-A. In contrast, JAM-A S285 is displaced from the cell–cell contacts to the cytoplasm at 30 min post-infection. Tyrosine phosphorylation of JAM-A Y280 is apparent at 60–120 min post-infection. Scale bars, 10 µm. ( B ) EPEC induces the endocytosis of occludin and ZO-1 at 1 and 2 h post-infection, respectively. Scale bars, 10 µm. ( C ) TER drops significantly as early as 45 min post-infection and progressively decreases over time as more TJ proteins are displaced. TER reported as percent change from baseline. * p < 0.01, *** p < 0.001.

Article Snippet: Par3 (07-330, EMD Millipore), Par6 (ab49776 and ab6022, Abcam, Cambridge, MA, USA), aPKCζ (sc-17781, Santa Cruz Biotechnology, Dallas, TX, USA), p-aPKCζ–T560 (ab62372, Abcam, Cambridge, MA, USA), p-aPKCζ–T410 (sc-12894R, Santa Cruz Biotechnology, Dallas, TX, USA), actin (A2066, Sigma-Aldrich, St. Louis, MO, USA), F-actin BODIPY 558/568 Phalloidin (B3475, Invitrogen, Life Technologies Carlsbad, CA, USA), occludin (33-1500, Invitrogen, Life Technologies, Carlsbad, CA, USA), JAM-A S285 (sc-17430, Santa Cruz Biotechnology, Dallas, TX, USA), JAM-A Y280 (600-401-GN5, Rockland Immunochemicals Inc, Limerick, PA), JAM-A (361700, Invitrogen, Life Technologies, Carlsbad, CA, USA), and ZO-1 (61-7300, Invitrogen, Life Technologies, Carlsbad, CA, USA).

Techniques: Infection, Phospho-proteomics