Journal: The FASEB Journal

Article Title: Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro‐inflammatory host‐microbe interactions in the colon

doi: 10.1096/fj.202101135r

Figure Lengend Snippet: FIGURE 1 A triple coculture model consisting of a collagen coating with confluent THP-1, LS-174T and T84 cells in DMEM/F-12 at the basolateral side, in indirect contact with apical microbes adhered to a solidified agar-mucin layer covering the Transwell polycarbonate membrane filter (0.4 µm pores) was developed to study host-microbe interactions in the human gut

Article Snippet: The T84 cell line (CLS 300354) and LS- 174T cell line (CLS 300392) were obtained from Cell Lines Service (Eppelheim, Germany).

Techniques: Membrane

Journal: The FASEB Journal

Article Title: Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro‐inflammatory host‐microbe interactions in the colon

doi: 10.1096/fj.202101135r

Figure Lengend Snippet: FIGURE 2 Triple cocultures morphologically organize in networks producing thicker and denser MUC2 layers than T84 monocultures. (A) F-actin, nuclei, MUC2 mucin staining and transmission image showing morphological characteristics of T84 monolayer (top) and triple coculture (bottom) cell models as visualized by confocal microscopy. Blue arrows: epithelial T84 cells; White arrows: LS-174T cells; Yellow arrows: Macrophage-like THP-1 cells; Green arrows: apoptotic structures. (B) The thickness of MUC2 layers in monolayers versus triple cocultures prior to microbial exposure (n = 6). (C) Example of a cross section and top view of the MUC2 layer formed on top of the monolayer and triple coculture host cells. *p = .0081

Article Snippet: The T84 cell line (CLS 300354) and LS- 174T cell line (CLS 300392) were obtained from Cell Lines Service (Eppelheim, Germany).

Techniques: Staining, Transmission Assay, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: shRNA-mediated XRCC2 gene knockdown efficiently sensitizes colon tumor cells to X-ray irradiation in vitro and in vivo.

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Figure 3. Knockdown of XRCC2 by shRNA inhibited cell growth of T84 cells. Cells were transfected with either shRNA-XRCC2 or shRNA-SC. The effect of XRCC2 suppression on cell growth in T84 cell line was examined by MTT assay. The values are presented as the mean ± SD (n = 12). * p < 0.05, ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, shRNA, Transfection, MTT Assay, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

doi: 10.1016/j.jcmgh.2022.01.003

Figure Lengend Snippet: Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling

Article Snippet: They were then transfected in serum-free medium using the T84 Cell Avalanche Transfection Reagent (EZ Biosystems, College Park, MD), prepared in complexes with plasmid DNA according to the manufacturer’s instructions (15 μL of T84 Cell Avalanche Transfection Reagent for 5 μg of total DNA: pRC/CMV, pRC/CMV HA-CK2α, and/or pRC/CMV Myc-CK2β).

Techniques: Binding Assay, Protein Binding

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

doi: 10.1016/j.jcmgh.2022.01.003

Figure Lengend Snippet: CK2 interacts with NOXO1 in T84 colon epithelial cells under inflammatory conditions. ( A ) Immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon T84 cells stimulated with TNFα (5 ng/mL) or IL17 (50 ng/mL) individually or in combination for 24 hours at 37°C. Representative of 3 independent experiments. ( B ) ROS production was measured by chemiluminescence in T84 cells stimulated as in (A) . n = 3. ( C ) Immunoblots of CK2 α/α′ in NOXO1 IPP from T84 cells stimulated as in (A) . Representative of 4 independent experiments. ( D ) Densitometry analysis of CK2 α/α′ in NOXO1 IPP as in ( C ), normalized to CK2 α/α′expression in cell lysates; n = 4; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05. ( E ) Confocal microcopy of T84 cells co-stimulated or not with TNFα + IL17 for 24 hours at 37°C. NOXO1 ( green ), CK2 α/α′ ( red ), DAPI ( blue ). Scale bar: 10 μm. Representative of 6 independent experiments.

Article Snippet: They were then transfected in serum-free medium using the T84 Cell Avalanche Transfection Reagent (EZ Biosystems, College Park, MD), prepared in complexes with plasmid DNA according to the manufacturer’s instructions (15 μL of T84 Cell Avalanche Transfection Reagent for 5 μg of total DNA: pRC/CMV, pRC/CMV HA-CK2α, and/or pRC/CMV Myc-CK2β).

Techniques: Western Blot, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

doi: 10.1016/j.jcmgh.2022.01.003

Figure Lengend Snippet: CK2 interacts directly with NOXO1 through the N-terminal region mostly containing the PX domain. ( A ) Coomassie Blue staining of recombinant NOXO1, p47 PHOX , NOXA1, and p67 PHOX . ( B ) Dot-blot analysis of interaction between CK2 and recombinant NOXO1, p47 PHOX , NOXA1, or p67 PHOX . Representative of 3 independent experiments. ( C ) Schematic representation and Coomassie Blue staining of GST fusion proteins of the NOXO1 full-length (β isoform), N-terminal (1-157), and C-terminal regions (232-371). ( D ) Pull-down of CK2 from resting T84 cells lysates by full-length GST-NOXO1, GST-NOXO1 (1-157), GST-NOXO1 (232-371), or GST (control). Representative of 3 independent experiments. ( E ) Densitometry analysis of CK2 α/α′ pull-downed as in ( D ), normalized to GST-fusion proteins; n = 3; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗∗∗ P < 0.0001.

Article Snippet: They were then transfected in serum-free medium using the T84 Cell Avalanche Transfection Reagent (EZ Biosystems, College Park, MD), prepared in complexes with plasmid DNA according to the manufacturer’s instructions (15 μL of T84 Cell Avalanche Transfection Reagent for 5 μg of total DNA: pRC/CMV, pRC/CMV HA-CK2α, and/or pRC/CMV Myc-CK2β).

Techniques: Staining, Recombinant, Dot Blot, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

doi: 10.1016/j.jcmgh.2022.01.003

Figure Lengend Snippet: Inhibition of CK2 enhances ROS production by NOX1 in colon T84 epithelial cells under inflammatory conditions. ( A ) ROS production was measured by chemiluminescence in T84 cells co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 after 24-hour incubation at 37°C. n = 3. ( B ) CK2 activity (top) assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody and NOXO1 expression (middle) in T84 cells co-stimulated as in (A) . Representative of 3 independent experiments. ( C ) Concentration-dependent effect of CX-4549 on ROS production by NOX1 in T84 cells co-stimulated as in (A) in presence or absence of various concentrations of CX-4945 for 24 hours at 37°C. Data were expressed as percentage of control (cells treated with TNFα + IL17 in absence of CX-4549); n = 7 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗ P < .01. ( D ) Concentration-dependent effect of TBBz on ROS production by NOX1 measured by chemiluminescence in T84 cells co-stimulated as in (A) in the presence or absence of various concentrations of TBBz for 24 hours at 37°C. ( E ) Concentration-dependent effect of CX4945 on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM; one-way ANOVA with Dunnett multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( F ) Concentration-dependent effect of TBBz on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM.

Article Snippet: They were then transfected in serum-free medium using the T84 Cell Avalanche Transfection Reagent (EZ Biosystems, College Park, MD), prepared in complexes with plasmid DNA according to the manufacturer’s instructions (15 μL of T84 Cell Avalanche Transfection Reagent for 5 μg of total DNA: pRC/CMV, pRC/CMV HA-CK2α, and/or pRC/CMV Myc-CK2β).

Techniques: Inhibition, Incubation, Activity Assay, Expressing, Concentration Assay, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

doi: 10.1016/j.jcmgh.2022.01.003

Figure Lengend Snippet: Effect of CK2β and CK2α overexpression on CK2 activity and ROS production in colon T84 epithelial cells. ( A ) Immunoblots of HA-CK2β and Myc-CK2α overexpressed individually in colon T84 cells. ( B ) CK2 activity assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody in T84 cells overexpressing CK2β or CK2α. ( C ) ROS production was measured by chemiluminescence in T84 cells overexpressing CK2β or CK2α. n = 3. ( D ) Effect of increasing concentrations of recombinant CK2β subunit on phosphorylation of NOXO1 by CK2 in vitro . Autoradiography (Autorad.) and Ponceau-Red are shown.

Article Snippet: They were then transfected in serum-free medium using the T84 Cell Avalanche Transfection Reagent (EZ Biosystems, College Park, MD), prepared in complexes with plasmid DNA according to the manufacturer’s instructions (15 μL of T84 Cell Avalanche Transfection Reagent for 5 μg of total DNA: pRC/CMV, pRC/CMV HA-CK2α, and/or pRC/CMV Myc-CK2β).

Techniques: Over Expression, Activity Assay, Western Blot, Recombinant, Phospho-proteomics, In Vitro, Autoradiography

Journal: BMJ Open Respiratory Research

Article Title: Differential response to bacteria, and TOLLIP expression, in the human respiratory tract

doi: 10.1136/bmjresp-2014-000046

Figure Lengend Snippet: TOLLIP expression in nasal and alveolar epithelium. (A) T84 cells were plated at two different cell densities: 5×10 5 per well (lanes 1, 2); 2×10 6 , (lanes 3, 4). Lane 5 represents a negative control without the reverse transcriptase. GAPDH was used as a housekeeping gene. (B) TOLLIP expression was quantified in primary nasal and alveolar epithelium. *p<0.05 by Mann-Whitney U test. (C and D) Cell lines were infected with Staphylococcus aureus strain Newman. RNA extraction was performed followed by RT-PCR. Panel C shows RPMI 2650 cells—and panel D A549 cells—infected with S. aureus . Lanes: (1) positive control for TOLLIP from cell line T84; (2 and 3) unstimulated; (4 and 5) cells with S. aureus at 1.1×10 5 cfu/mL; (6 and 7) cells with S. aureus at 1.6×10 5 cfu/mL. GAPDH was used as a housekeeping gene. Band size for TOLLIP 347 bp and for GAPDH 727 bp (TOLLIP, Toll-interacting protein; RT-PCR, reverse transcriptase PCR).

Article Snippet: The human colonic carcinoma cell line T84 and the human nasal carcinoma cell line RPMI 2650 were from LGC Promochem (Manassas, Virginia, USA; ATCC numbers CCL-248 and CCL-30 respectively).

Techniques: Expressing, Negative Control, Reverse Transcription, MANN-WHITNEY, Infection, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Positive Control

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