intestinal epithelial t84 cell line  (ATCC)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer

doi: 10.3389/fcimb.2022.824547

Figure Lengend Snippet: Changes in barrier function due to infection of various A. sobria strains to the apical side of the T84 intestinal epithelial monolayer. (A) T84 cells were grown on a Transwell system and then infected with several A. sobria strains. After 9 hr of infection (MOI = 5) to the apical side of the T84 monolayer, the TER was measured (post-infection). The obtained TER value was compared with the TER value before infection (pre-infection). NT: The TER value was measured without bacterial infection. The experiments were performed in triplicate. The data are mean ± SD (error bars). We performed an ANOVA analysis with Dunnett’s test and showed significant differences (*p < 0.05) between the data obtained from each bacterial strain and the control data (NT). (B) The ability of the A. sobria strains to translocate across the T84 intestinal epithelial monolayer at 9 hr after infection (MOI = 5) was evaluated by calculating the colony forming units per mL (CFU/mL) of the culture medium in the lower chambers (the basal side of the T84 monolayer). The number of bacteria present in the solution (10 μL) of the lower chamber was calculated by the plating method. In this method, the number of bacteria less than 100 CFU/mL cannot be determined. If no bacteria were found in 10 μL of solution in the lower chamber, it was displayed as “<10 2 ” in the figure. The experiments were performed in triplicate. The data are mean ± SD (error bars). We performed an ANOVA analysis with Dunnett’s test and showed significant differences (*p < 0.05) between the data obtained from each bacterial strain and the control data (NT). (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. ND: The proteolytic activity could not be sufficiently detected in this experimental condition. 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. The arrow indicates the position of the ASP (65-kDa) band fractionated by SDS-PAGE.

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

Techniques: Infection, Control, Bacteria, Activity Assay, Western Blot, SDS Page

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer

doi: 10.3389/fcimb.2022.824547

Figure Lengend Snippet: Degradation of the protein components constituting TJs due to infection of various A. sobria strains to the apical side of the T84 intestinal epithelial monolayer. T84 cells were cultured in 12-well microplates and then infected with several A. sobria strains. After 2 hr of infection (MOI = 5) to the apical side of the T84 monolayer, the cell extracts were prepared. The protein components constituting TJs (ZO-1, ZO-2, ZO-3, occludin, and claudin-1, -3, -4, and -7) were detected by using a specific antibody against each protein. The results of the quantitative analysis of the relative ratio (%) of the amount of blotted protein (ZO-1, ZO-2, and ZO-3) to the amount of GAPDH are shown below the western blotting image. Concerning the degradation of claudin-7, the results of the quantitative analysis of the relative ratio (%) of the amount of the blotted claudin-7 degradation fragment (Claudin-7 FG) to the amount of GAPDH are shown. These experiments were performed in triplicate. The data are mean ± SD (error bars). We performed an ANOVA analysis with Dunnett’s test and showed significant differences (*p < 0.05, **p < 0.01) between the data obtained from each bacterial strain and the control data (NT).

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

Techniques: Infection, Cell Culture, Western Blot, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer

doi: 10.3389/fcimb.2022.824547

Figure Lengend Snippet: Effects of various protease inhibitors on the degradation of ZO-1, ZO-2, ZO-3, and claudin-7 due to infection of A. sobria strain 288 to the apical side of the T84 intestinal epithelial monolayer. T84 cells were cultured in 12-well microplates and then infected with A. sobria strain 288 in the presence of the protease inhibitors shown. After 2 hr of infection (MOI = 5) to the apical side of the T84 monolayer, the cell extracts were prepared. The protein components (ZO-1, ZO-2, ZO-3, and claudin-7) constituting TJs were detected by using a specific antibody against each protein. The results of the quantitative analysis of the relative ratio (%) of the amount of blotted protein (ZO-1, ZO-2, and ZO-3) to the amount of GAPDH are shown below the western blotting image. Concerning the degradation of claudin-7, the results of the quantitative analysis of the relative ratio (%) of the amount of the blotted Claudin-7 FG to the amount of GAPDH is shown. These experiments were performed in triplicate. The data are mean ± SD (error bars). *p < 0.05; significance was tested by comparison with the control data (NT; without infection with strain 288).

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

Techniques: Infection, Cell Culture, Western Blot, Comparison, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer

doi: 10.3389/fcimb.2022.824547

Figure Lengend Snippet: Effect of the asp gene disruption on the degradation of the protein components constituting TJs. (A) T84 cells were cultured in 12-well microplates and then infected with A. sobria strain 288 and its derivative strains shown in the figure. After 2 hr of infection (MOI = 5) to the apical side of the T84 monolayer, the cell extracts were prepared. The protein components (ZO-1, ZO-2, ZO-3, and claudin-7) constituting TJs were detected by using a specific antibody against each protein. The results of the quantitative analysis of the relative ratio (%) of the amount of blotted protein (ZO-1, ZO-2, and ZO-3) to the amount of GAPDH are shown below the western blotting image. Concerning the degradation of claudin-7, the results of the quantitative analysis of the relative ratio (%) of the amount of the blotted Claudin-7 FG to the amount of GAPDH are shown. These experiments were performed in triplicate. The data are mean ± SD (error bars). *p < 0.01; significance was tested by comparison with the control data (NT). (B) 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. ND: The proteolytic activity could not be sufficiently detected in this experimental condition. The data are mean ± SD (error bars). (C) The presence of ASP in the culture supernatant of each strain was immunologically detected by a western blotting analysis as described in the text. The arrow indicates the position of the ASP (65-kDa) band fractionated by SDS-PAGE.

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

Techniques: Disruption, Cell Culture, Infection, Western Blot, Comparison, Control, Activity Assay, SDS Page

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer

doi: 10.3389/fcimb.2022.824547

Figure Lengend Snippet: Effect of the purified ASP on various protein components constituting TJs. T84 cells were cultured in 12-well microplates and then treated with various concentrations (nM) of the purified ASP. For the evaluation of the effect of the serine protease inhibitor, T84 cells were also treated with 500 nM of the purified ASP in the presence of PMSF. After treatment for 2 hr, the cell extracts were prepared. The protein components (ZO-1, ZO-2, ZO-3, and claudin-7) constituting TJs were detected by using a specific antibody against each protein. The results of the quantitative analysis of the relative ratio (%) of the amount of blotted protein (ZO-1, ZO-2, and ZO-3) to the amount of GAPDH are shown below the western blotting image. Regarding the degradation of claudin-7, the results of the quantitative analysis of the relative ratio (%) of the amount of the blotted Claudin-7 FG to the amount of GAPDH are shown. These experiments were performed in triplicate. The data are mean ± SD (error bars). *p < 0.01; significance was tested by comparison with the control data (0 nM ASP).

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

Techniques: Purification, Cell Culture, Protease Inhibitor, Western Blot, Comparison, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer

doi: 10.3389/fcimb.2022.824547

Figure Lengend Snippet: Degradation of ZO-1, ZO-2, ZO-3, and claudin-7 due to infection of the asp -deficient A. sobria strain (288 Δ asp ) to the apical side of the T84 intestinal epithelial monolayer in the presence of various concentrations of the purified ASP. T84 cells were cultured in 12-well microplates and then infected with the A. sobria 288 Δ asp strain in the presence of various concentrations of the purified ASP. After 2-hr treatment, the cell extracts were prepared. ZO-1, ZO-2, ZO-3, and claudin-7 were detected by using a specific antibody against each protein. The results of the quantitative analysis of the relative ratio (%) of the amount of blotted protein (ZO-1, ZO-2, and ZO-3) to the amount of GAPDH are shown below the western blotting image. Concerning the degradation of claudin-7, the results of the quantitative analysis of the relative ratio (%) of the amount of the blotted Claudin-7 FG to the amount of GAPDH are shown. These experiments were performed in triplicate. The data are mean ± SD (error bars). *p < 0.01; significance was tested by comparison with the control data (NT).

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

Techniques: Infection, Purification, Cell Culture, Western Blot, Comparison, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer

doi: 10.3389/fcimb.2022.824547

Figure Lengend Snippet: Confocal images of the T84 intestinal epithelial monolayer infected with A. sobria strain 288 and its derivative strains. T84 cells were grown on a Transwell system and then infected with A. sobria strain 288 and its derivative strains, A. sobria strain 288 Δ asp and A. sobria strain 288 Δ asp :: asp . ZO-1 ( green fluorescence ) and claudin-7 ( red fluorescence ) were immunologically observed. Intracellular DNA was also immunologically detected ( blue fluorescence ). x-y: The x-y image of the T84 intestinal epithelial monolayer surface. x-z: Cross-sectional image of the deeper layer. White bars: 10 μm.

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

Techniques: Infection, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Beneficial Effects of Natural Mineral Waters on Intestinal Inflammation and the Mucosa-Associated Microbiota

doi: 10.3390/ijms22094336

Figure Lengend Snippet: Secretion of the pro-inflammatory cytokine IL-8 by T84 cells after stimulation with an LF82 bacterial lysate ( A ) or exposure to H 2 O 2 ( B ) in the presence of 50 µL of different waters (distilled water or NMW1 or NMW2). The results are presented as the mean ± SEM of at least five independent experiments. Statistical comparisons were carried out by one-way ANOVA with the Bonferroni post hoc test (** p < 0.01, *** p < 0.001, **** p < 0.0001, ns—not significant) after normality testing using the Kolmogorov–Smirnov test.

Article Snippet: The human intestinal epithelial cell line T84 was obtained from the American Type Culture Collection (ATCC ® CCL-248 TM , Manassas, VA, USA) and maintained in an atmosphere containing 5% CO 2 at 37 °C in the culture medium recommended by ATCC.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Beneficial Effects of Natural Mineral Waters on Intestinal Inflammation and the Mucosa-Associated Microbiota

doi: 10.3390/ijms22094336

Figure Lengend Snippet: Histological grading of intestinal inflammation.

Article Snippet: The human intestinal epithelial cell line T84 was obtained from the American Type Culture Collection (ATCC ® CCL-248 TM , Manassas, VA, USA) and maintained in an atmosphere containing 5% CO 2 at 37 °C in the culture medium recommended by ATCC.

Techniques: