Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: T84 cells were cultured in a Transwell system and then infected with several types of A . sobria strains. After 3 hr of infection (MOI = 5), the reduction in TER was measured. The TER value at 0 hr of infection was taken as 100%. NT: The TER value was measured without bacterial infection. The experiments were performed in triplicate. The data are mean ± SD (error bars). *p<0.01. B: The ability of the A . sobria strains to translocate across the intestinal epithelial cells (T84 cells) at 6 hr after infection (MOI = 5) was assessed using quantitative cultures of medium obtained from the lower chambers. NT: The experiment was done without bacterial infection. ND: The bacterial translocation could not be detected in this experimental condition. The experiments were performed in triplicate. The data are mean ± SD (error bars). C: The proteolytic activity in the culture supernatant of each A . sobria strain was measured as described in the text. The experiments were performed in triplicate. The data are mean ± SD (error bars). D: The presence of ASP in the culture supernatant of each strain was immunologically detected by a western blotting analysis as described in the text.

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Cell Culture, Infection, Translocation Assay, Activity Assay, Western Blot

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: The asp gene of A . sobria 288 strain was knocked out. The immunological analysis using western blotting revealed that the asp -knocked-out strain (#288 ΔASP) did not produce ASP and the complemented strain (#288 ΔASP::ASP) produced ASP again. B: The proteolytic activity in the culture supernatant of each strain. The experiments were performed in triplicate. The data are mean ± SD (error bars). C: T84 cells were cultured in a Transwell system and then infected with the wild-type A . sobria strain (#288), the asp -knocked-out strain (#288 ΔASP), or the complemented strain (#288 ΔASP::ASP). After 6 hr of infection (MOI = 5), the ability of these A . sobria strains to translocate across the T84 cell monolayer was assessed in the same way as that described in the legend. The experiments were performed in triplicate. The data are mean ± SD (error bars). *p<0.01, **p<0.05.

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Western Blot, Produced, Activity Assay, Cell Culture, Infection

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: T84 cells were cultured in a Transwell system, and the TER value was measured in the presence of various concentrations (nM) of ASP or absence (NT) of ASP. We also examined the effect of the serine protease inhibitor PMSF on the action of ASP. B: The passive diffusion of FITC-labeled dextran molecules across the T84 monolayer (from the apical side to the basolateral side) treated with ASP was measured. All experiments were performed in triplicate. The data are mean ± SD (error bars).

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Cell Culture, Protease Inhibitor, Diffusion-based Assay, Labeling

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: T84 cells were treated with (nM) or without (NT) various concentrations of ASP before the extraction. After the extraction, we detected the proteins constituting the junctional complexes by using a specific antibody against each protein shown in the figure. The results of quantitative analysis of the amount of blotted protein are also shown below the image of western blotting. These experiments were performed in triplicate. The data are mean ± SD (error bars).

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Extraction, Western Blot

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: T84 cells were treated with or without (NT) 500 nM ASP for 6 hr. The cells were reacted with a specific antibody against nectin-1, nectin-2, and E-cadherin and visualized using the secondary antibody conjugated with a fluorescent substance, Cy5 or FITC. Nuclei were stained with PI. The merged images are shown in each panel. Merge 1: nectin-2 and E-cadherin, Merge 2: afadin and E-cadherin, and Merge 3: nectin-1 and E-cadherin. Z: Z-stack showing entire sample volume image was also shown.

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Staining

Journal: PLoS ONE

Article Title: Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

doi: 10.1371/journal.pone.0221344

Figure Lengend Snippet: A: T84 cells were infected (MOI = 1) with or without (NT) A . sobria strain 120, 123 or 288. Bacterial internalization was confirmed using the Aeromonas -specific probe FITC-AER66 as described in Materials and Methods ( fluorescence image ). The figure shown as ‘Merge’ is a superposition of the fluorescence image ( left side ) and the blight image ( center ). B: T84 cells were infected (MOI = 1) with A . sobria strain 120, 123 or 288. The number of bacteria survived from gentamicin protection assay was determined and indicated as colony forming unit (CFU). The experiments were performed in triplicate and the data are means ± SD (error bars).

Article Snippet: The intestinal epithelial cell line T84 was obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Infection, Fluorescence, Bacteria