Journal: Journal of Biological Chemistry

Article Title: A Cholera Toxin B-subunit Variant That Binds Ganglioside GM1 but Fails to Induce Toxicity

doi: 10.1074/jbc.m104245200

Figure Lengend Snippet: FIG. 1. Mutant CT-H57A fails to induce a secretory response from T84 cells. Shown is a time course of electrogenic Cl secretion (Isc) induced by the addition of 20 nM wt CT (G) or mutant CT-H57A (E) to the apical (A) or basolateral (B) surface of T84 cell monolayers (with data points representing the means S.D. of two independent mono- layers). Three independent experiments gave similar results. In C, the dose response induced by wt CT (G) or CT-H57A (E) was measured as electrogenic Cl secretion (Isc) induced 110 min after the addition of toxin to the apical surfaces of polarized T84 cells. D shows that the Cl

Article Snippet: T84 cells obtained from American Type Culture Collection were cultured and passaged as described previously (16).

Techniques: Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: A Cholera Toxin B-subunit Variant That Binds Ganglioside GM1 but Fails to Induce Toxicity

doi: 10.1074/jbc.m104245200

Figure Lengend Snippet: FIG. 2. Mutant CTB-H57A fails to compete with CT for induc- tion of Cl secretion. The secretory response (Isc) induced by 0.5 nM CT applied apically to polarized T84 cells was measured in the presence or absence of 500 nM competitor CTB (A) or CTB-H57A (B). Data points represent the means S.D. of two independent monolayers, with four independent experiments giving similar results.

Article Snippet: T84 cells obtained from American Type Culture Collection were cultured and passaged as described previously (16).

Techniques: Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: A Cholera Toxin B-subunit Variant That Binds Ganglioside GM1 but Fails to Induce Toxicity

doi: 10.1074/jbc.m104245200

Figure Lengend Snippet: FIG. 3. Binding of CTB-H57A to T84 cell membranes. A, steady- state binding of wt CTB (G) and CTB-H57A (E) to the cell-surface membranes of T84 cells at 4 °C by modified ELISA, with data points representing the means S.E. of four independent monolayers. Solid lines represent curve fits to a mass action model for binding a single class of receptors. The dashed line represents curve fit to a mass action model for binding two classes of receptors. Three independent experi- ments gave similar results. B, binding of 4 nM HRP-labeled CTB com- peted with increasing concentrations of wt CTB (G) or CTB-H57A (E) on the cell surface of T84 cells at 4 °C.

Article Snippet: T84 cells obtained from American Type Culture Collection were cultured and passaged as described previously (16).

Techniques: Binding Assay, Modification, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Journal of Biological Chemistry

Article Title: A Cholera Toxin B-subunit Variant That Binds Ganglioside GM1 but Fails to Induce Toxicity

doi: 10.1074/jbc.m104245200

Figure Lengend Snippet: FIG. 5. Association of toxinGM1 complexes with detergent-in- soluble membrane microdomains. A, Western blot for the toxin B-subunit contained in the lipid rafts or detergent-soluble fractions (Sol) of T84 cell monolayers exposed to 20 nM wt CTB or CTB-H57A; B, densitometry of three independent experiments as described in A (means S.D.).

Article Snippet: T84 cells obtained from American Type Culture Collection were cultured and passaged as described previously (16).

Techniques: Membrane, Western Blot

Journal: Journal of Biological Chemistry

Article Title: A Cholera Toxin B-subunit Variant That Binds Ganglioside GM1 but Fails to Induce Toxicity

doi: 10.1074/jbc.m104245200

Figure Lengend Snippet: FIG. 6. CTB-H57A fails to enter T84 cells. A, rate of endocytosis for wt CTB (G) and CTB-H57A (E) measured as the disappearance of cell surface-bound toxin. T84 cells preincubated at 4 °C with 20 nM concentrations of the respective toxins were moved to 37 °C for the indicated times, and the specific surface-exposed toxin was measured by modified ELISA. B, total cell-associated CTB before and after endocytosis as assessed by Western blotting of total cell lysates. Lanes labeled H57A(3X) were loaded with 3-fold more cell extracts. m, minutes. C, immunopre- cipitation of CTB from total T84 cell ex- tracts before and after endocytosis and co-immunoprecipitation of the CT B-sub- unit with ganglioside GM1. Toxin B-sub- units were detected by Western blotting, and GM1 was detected by ligand blotting using HRP-labeled CTB as ligand. D, sul- fation of wt CTB and CTB-H57A contain- ing the sulfation motif SAEDYEYPS ap- pended to the C terminus of the B-subunit after 50 min of incubation with T84 cells at 37 °C. The lower panel shows that equal amounts of cell-associated toxin were loaded in each lane after immuno- precipitation from total T84 cell extracts. Immunoprecipitation of CTB-H57A was performed on a 3-fold greater mass of T84 cell lysates.

Article Snippet: T84 cells obtained from American Type Culture Collection were cultured and passaged as described previously (16).

Techniques: Modification, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Immunoprecipitation, Incubation

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: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Immunostaining of T84 cell monolayers for (A) occludin and (B) ZO‐1 showing the formation of a continuous monolayer of cells. (C, D) mRNA levels of CYP1A1 and CYP1B1 cultured in 24‐well plates (‘24w’) and as monolayers on Transwell inserts. The individual ΔCt values (the difference in threshold cycle between the mRNA of the CYP and the housekeeper mRNA [RPL19]) are shown with the means represented by the bars. The right Y‐axes show data where the mean for the 24‐well plates is set to 100%.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Immunostaining, Cell Culture

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effect of IFNγ treatment of T84 cells upon their oxylipin content (fmol per well)

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Control

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effects of IFNγ and TMS upon the mRNA expression of HIF1, AhR and proteins involved in the barrier function of T84 cells

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Expressing, Control

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Panel A, effects of vehicle (V, N = 21 and 20 for control and IFNγ‐treated respectively), and TMS (T, 100 nM; N = 13 and 19 for control and IFNγ‐treated respectively) treatment upon TEER levels in T84 cells cultured in Transwells. Vehicle or IFNγ (10 ng·mL−1) was added basolaterally and vehicle or TMS apically prior to culture for 24 h. In all cases the values shown are the TEER values at 24 h expressed as a percentage of the corresponding values for the same conditions at t = 0. As the sample sizes are different, we calculated ANOVA P values using a permutation test (function aovp in the package lmPerm for R). The P values for IFNγ, TMS and IFNγ × TMS were all <0.05. Pairwise P values (after controlling for α inflation using a Bonferroni correction) were calculated using two‐sided permutation tests using the function permTS in the package perm for R and are shown in the Figure: *P < 0.05; significantly different as indicated; NS, not significantly different. Panel B, % reversal of the effect of IFNγ upon TEER by TMS (N = 19), benzo[a]pyrene (Benzo, 10 μM, N = 6)) and α‐naphthoflavone (α‐Nap, 100 nM, N = 6). For each experiment, % reversal was calculated as [100 – (100 × (ΔTEERcpd‐con − ΔTEERcon)/(ΔTEERifn − ΔTEERcon)], where ‘ΔTEER’ refers to the change (in Ω·cm2 seen between t0 and t24 for the different conditions; ‘cpd’ refers to the combination of test compound and IFNγ treatment, ‘con’ to the untreated cells and ‘ifn’ to cells treated with IFNγ and vehicle. A one‐way permutation ANOVA (independence_test in the package coin for R) gave a P value <0.05. One sample t‐tests of each condition versus 0% gave a significant P value for TMS, but not for benzo[a]pyrene or α‐naphthoflavone.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Control, Cell Culture

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effects of vehicle (v) and TMS (T, 100 nM) upon the mRNA for A, IL8 and B, TNFα (N = 6, except for the TMS‐treated control cells, where N = 5) in control and IFNγ‐treated cells. The T84 cells in Transwells were treated as described in Figure 4. For the IFNγ‐treated cells, the mean values for vehicle‐ and TMS‐treated cells were not significantly different.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Control

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effect of IFNγ treatment upon the properties of T84 cells cultured in monolayers on Transwells. IFNγ (10 ng·mL−1) was added basolaterally at t0 and the cells were cultured for 24 h. Panel A, TEER values for the controls (N = 31) and IFNγ‐treated (N = 30) cells are pooled from several experimental series. In all the scatterplots presented in this paper, the solid bars show the mean values. A two‐way type 3 ANOVA with time as repeated measure gave F 1,59 values for time, IFNγ and the interaction time × IFNγ of 140, 10.0 and 235, respectively (all statistically significant); *P < 0.05; significantly different as indicated; Sidak's multiple comparisons test. Residual plots (of a corresponding linear mixed model analysis) indicated no obvious heteroscedasticity or non‐normality of the distribution. Panel B, mRNA levels for CYP1A1 (N = 10), CYP1B1 (N = 10), IL8 (N = 8) and TNFα (N = 8) following 24 h of treatment with either vehicle or IFNγ. *P < 0.05; significantly different as indicated; two‐tailed t‐tests.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Cell Culture, Two Tailed Test

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effect of PDTC (100 μM) and YC‐1 (50 μM) upon mRNA expression of (A) CYP1A1 and (B) CYP1B1 for IFNγ‐treated T84 cells (N = 6). Cells were seeded overnight in 24‐well plates and incubated with vehicle or the compounds for 1 h, before incubation with IFNγ (10 ng·mL−1) for 8 h. *P < 0.05; significantly different from control (concomitantly cultured cells in the absence of IFNγ); two‐tailed paired t‐test. For the IFNγ‐treated cells, Levene's test for homogeneity of variance was significant for CYP1B1 and so, we analysed the data using a two‐way permutation test (function aovp in the package lmperm for R). For CYP1A1, a significant P value was found for the main effect of YC‐1. For CYP1B1, significant P values were found for YC‐1 and the interaction PDTC × YC‐1.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Expressing, Incubation, Control, Cell Culture, Two Tailed Test

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: 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