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

Journal: Oncotarget

Article Title: Hypermethylation of NDN promotes cell proliferation by activating the Wnt signaling pathway in colorectal cancer

doi: 10.18632/oncotarget.17580

Figure Lengend Snippet: (A) Immunostaining of NDN protein in CRC tissue samples and normal colorectal tissues. (B) Expression analyses of NDN protein in 12 surgical CRC tissues and the paired normal intestine epithelial samples using Western blot. β-tubulin was used as a loading control. (C) qRT-PCR analysis of NDN expression in 21 paired colorectal cancer tissues; NDN was quantified relative to the matched adjacent no tumor tissues. Error bars represent means ± SD calculated from three parallel experiments. (D) Expression analyses of NDN protein and mRNA in the normal intestinal epithelial cell FHC and five CRC cell lines through Western blot and qRT-PCR. Each bar represents the mean ± SD of three parallel replicates. (E) Influence of NDN expression on overall survival through Kaplan-Meier analysis in 84 CRC patients.

Article Snippet: Human normal intestinal epithelial cell FHC and five CRC cell lines SW480, HT29, HCT116, LS174T, and RKO were obtained from the The Global Bioresource Center (ATCC, USA).

Techniques: Immunostaining, Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Molecular Medicine Reports

Article Title: Atractylenolide III inhibits epithelial-mesenchymal transition in small intestine epithelial cells by activating the AMPK signaling pathway

doi: 10.3892/mmr.2022.12614

Figure Lengend Snippet: Effect of ATL-III on the activity of IEC-6 cells. (A) Chemical structure of ATL-III. (B) Effect of ATL-III on the activity of IEC-6 cells was detected using an MTT assay. ATL-III, atractylenolide III.

Article Snippet: IEC-6 (rat small intestinal epithelial) cells were purchased from Merck-KGaA.

Techniques: Activity Assay, MTT Assay

Journal: Molecular Medicine Reports

Article Title: Atractylenolide III inhibits epithelial-mesenchymal transition in small intestine epithelial cells by activating the AMPK signaling pathway

doi: 10.3892/mmr.2022.12614

Figure Lengend Snippet: ATL-III inhibits the invasion and migration of IEC-6 cells induced by TGF-β1. (A) Cell morphology after TGF-β1 treatment was observed under a microscope. (B) Cell morphology after ATL-III treatment was observed under a microscope. Magnification, ×200. (C) Migration of IEC-6 cells was detected using a wound healing assay. (D) Cell invasion was detected using a Transwell assay. Histograms of cell (E) migration and (F) invasion rates. (G) Expression level of MMP9, as detected via western blotting. Magnification, ×100. ***P<0.001 vs. control group; ## P<0.01, ### P<0.001 vs. TGF-β1 group; n≥3. ATL-III, atractylenolide III.

Article Snippet: IEC-6 (rat small intestinal epithelial) cells were purchased from Merck-KGaA.

Techniques: Migration, Microscopy, Wound Healing Assay, Transwell Assay, Expressing, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Atractylenolide III inhibits epithelial-mesenchymal transition in small intestine epithelial cells by activating the AMPK signaling pathway

doi: 10.3892/mmr.2022.12614

Figure Lengend Snippet: ATL-III inhibits the EMT of IEC-6 cells induced by TGF-β1. (A) Expression levels of protein associated with EMT, as detected via western blotting. (B) Expression levels of N-cadherin and (C) E-cadherin were detected using an immunofluorescence assay. Magnification, ×200. ***P<0.001 vs. control group; ## P<0.01, ### P<0.001 vs. TGF-β1 group; n≥3. ATL-III, atractylenolide III; EMT, epithelial-mesenchymal transition; ZO-1, zonula occludens-1.

Article Snippet: IEC-6 (rat small intestinal epithelial) cells were purchased from Merck-KGaA.

Techniques: Expressing, Western Blot, Immunofluorescence, Control

Journal: Molecular Medicine Reports

Article Title: Atractylenolide III inhibits epithelial-mesenchymal transition in small intestine epithelial cells by activating the AMPK signaling pathway

doi: 10.3892/mmr.2022.12614

Figure Lengend Snippet: ATL-III activates the AMPK signaling pathway in IEC-6 cells. (A) Expression levels of p-AMPK and AMPK in the groups treated with different concentrations of ATL-III were measured using western blotting. (B) Expression levels of p-AMPK and AMPK in the groups treated with TGF-β1 and different concentrations of ATL-III were measured using western blotting. (C) Chemical structure of compound C. (D) Expression levels of p-AMPK and AMPK in the groups treated with TGF-β1, ATL-III and compound C were detected using western blotting. *P<0.05, ***P<0.001 vs. control group; ### P<0.001 vs. TGF-β1 group; △△P<0.01 vs. TGF-β1 + ATL-III (20 µmol/l) group; n≥3. ATL-III, atractylenolide III; AMPK, AMP-activated protein kinase; p-, phosphorylated; t-, total.

Article Snippet: IEC-6 (rat small intestinal epithelial) cells were purchased from Merck-KGaA.

Techniques: Expressing, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Atractylenolide III inhibits epithelial-mesenchymal transition in small intestine epithelial cells by activating the AMPK signaling pathway

doi: 10.3892/mmr.2022.12614

Figure Lengend Snippet: ATL-III inhibits the invasion, migration and EMT process of IEC-6 cells induced by TGF-β1 by activating the AMPK signaling pathway. (A) Cell migration, as determined using a wound healing assay. (B) Cells invasion was detected using a Transwell assay. Magnification, ×100. Histograms of cell (C) migration and (D) invasion rates. (E) Expression levels of MMP9 and proteins associated with EMT, as detected via western blotting. (F) N-cadherin and (G) E-cadherin expression was detected using an immunofluorescence assay. Magnification, ×200. ***P<0.001 vs. control group; ### P<0.001 vs. TGF-β1 group; △P<0.05, △△P<0.01 vs. TGF-β1 + ATL-III (20 µmol/l) group; n≥3. ATL-III, atractylenolide III; EMT, epithelial-mesenchymal transition; AMPK, AMP-activated protein kinase; ZO-1, zonula occludens-1.

Article Snippet: IEC-6 (rat small intestinal epithelial) cells were purchased from Merck-KGaA.

Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Western Blot, Immunofluorescence, Control