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: 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: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: qRT-PCR showing expression of human (A) CHOP , (B) spliced XBP1 [ XBP1(s) ], (C) ATF6 and (D) BiP in T84 cells infected with C. jejuni 11168H, 81-176 or 488 wild-type strains for 6 or 24 h at 37°C in a 5% CO 2 incubator (MOI of 200:1). T84 cells were treated with thapsigargin as a positive control. GAPDH was used as an internal control. One sample t -test was performed. Asterisks denote a statistically significant difference (** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Quantitative RT-PCR, Expressing, Infection, Positive Control, Control

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: Western blotting showing protein levels of human CHOP and both spliced & unspliced forms of XBP1 [XBP1(u) & XBP1(s)] in T84 (left panels) and Caco-2 cells (right panels) infected with C. jejuni 11168H, 81-176 or 488 wild-type strains for 24 h at 37°C in a 5% CO 2 incubator (MOI of 200:1). T84 cells were treated with thapsigargin (Tg) as a positive control. GAPDH was used as an internal control.

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Western Blot, Infection, Positive Control, Control

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: T84 or Caco-2 cells were pre-treated with 2 µM of thapsigargin for 6 h and infected with C. jejuni 11168H, 81-176 or 488 wild-type strains for 3 h at 37°C in a 5% CO 2 atmosphere (MOI of 200:1). For interaction assays, (A) T84 or (B) Caco-2 cells were washed with PBS, lysed with 0.1% (v/v) Triton X-100 and CFU/ml were recorded after incubation. For invasion assays, after infection with C. jejuni , (C) T84 or (D) Caco-2 cells were incubated with gentamicin (150 µg/ml) for 2 h to kill extracellular bacteria, then lysed with 0.1% (v/v) Triton X-100 and CFU/ml were recorded after incubation. For intracellular survival assays, the 2 h gentamicin treatment as for invasion assays was followed by further incubation with gentamicin (10 µg/ml) for 18 h, then (E) T84 or (F) Caco-2 cells were lysed with 0.1% (v/v) Triton X-100 and CFU/ml were recorded after incubation. One sample t -test was performed. Asterisks denote a statistically significant difference (** = p < 0.01; *** = p < 0.001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Infection, Incubation, Bacteria

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: (A) T84 or (B) Caco-2 cells were pre-treated with 3 µM of KIRA6, 100 µM of STF-083010 or 3 µM of GSK2656157 for 4 h at 37°C in a 5% CO 2 incubator. T84 and Caco-2 cells were then infected with C. jejuni 11168H, 81-176 or 488 wild-type strains for 24 h at 37°C in a 5% CO 2 incubator (MOI of 200:1). Human IECs were further treated with 3 µM of KIRA6 and 3 µM of GSK2656157 during C. jejuni infection. T84 and Caco-2 cells were washed with PBS and incubated with gentamicin (150 µg/ml) for 2 h to kill extracellular bacteria and then lysed with 0.1% (v/v) Triton X-100 and CFU/ml were recorded after incubation. One sample t -test was performed. Asterisks denote a statistically significant difference (* = p < 0.05; ** = p < 0.01).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Infection, Incubation, Bacteria

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: qRT-PCR showing expression of human (A, C) CHOP and (B, D) spliced XBP1 [ XBP1(s) ] in T84 or Caco-2 cells infected with either C. jejuni 11168H, 81-176 or 488 wild-type strains (MOI of 200:1) or treated with 2 µM of thapsigargin for 24 h at 37°C in a 5% CO 2 atmosphere. Prior to C. jejuni infection or thapsigargin treatment, T84 and Caco-2 cells were treated with 3 µM of KIRA6, 100 µM of STF-083010 or 3 µM of GSK2656157 for 4 h at 37°C in a 5% CO 2 incubator. T84 and Caco-2 cells were further treated with 3 µM of KIRA6 and 3 µM of GSK2656157 during C. jejuni infection or thapsigargin treatment. GAPDH was used as an internal control. Three biological and three technical replicates were performed for each experiment. Asterisks denote a statistically significant difference (* = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Quantitative RT-PCR, Expressing, Infection, Control

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: Western blotting showing protein levels of both spliced & unspliced forms of XBP1 [XBP1(u) & XBP1(s)] and CHOP in T84 cells infected with C. jejuni 11168H, 81-176 or 488 wild-type strains (MOI of 200:1) or treated with 2 µM of thapsigargin (Tg) for 24 h at 37°C in a 5% CO 2 incubator. Prior to C. jejuni infection or thapsigargin treatment, T84 and Caco-2 cells were pre-treated with 3 µM of KIRA6, 100 µM of STF-083010 or 3 µM of GSK2656157 for 4 h at 37°C in a 5% CO 2 incubator. T84 and Caco-2 cells were further treated with 3 µM of KIRA6 and 3 µM of GSK2656157 during C. jejuni infection or thapsigargin treatment. GAPDH was used as an internal control.

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Western Blot, Infection, Control

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: (A) T84 or (B) Caco-2 cells grown in a 96-well plate were treated with 3 µM of KIRA6, 3 µM of GSK2656157 or 30 µM of ML130 for 24 h or treated with 2 µM of thapsigargin for 6 h, 100 µM of STF-083010 for 4 h or 10 µM of BAPTA-AM for 1 h at 37°C in a 5% CO 2 incubator. Medium from each well was then analysed using a LDH assay. Cytotoxicity (%) was calculated by using the following equation: ( C. jejuni infected LDH activity – spontaneous LDH activity) / (maximum LDH activity – spontaneous LDH activity) x 100. Three biological and three technical replicates were performed for each experiment. Asterisks denote a statistically significant difference (* = p < 0.05; ** = p < 0.01).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Lactate Dehydrogenase Assay, Infection, Activity Assay

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: (A) T84 cells in a 24-well plate were infected with C. jejuni 11168H, 81-176 or 488 wild-type strains (MOI 200:1) or treated with 2 µM of thapsigargin for 6 or 24 h at 37°C in a 5% CO 2 incubator. T84 cells were pre-treated with 3 µM of KIRA6, 100 µM of STF-083010 or 3 µM of GSK2656157 for 4 h at 37°C in a 5% CO 2 incubator. T84 cells were then infected with C. jejuni 11168H, 81-176 or 488 wild-type strains (MOI 200:1) or treated with 2 µM of thapsigargin for 24 h at 37°C in a 5% CO 2 incubator. T84 cells were further treated with 3 µM of KIRA6 and 3 µM of GSK2656157 during C. jejuni infection. T84 cells in a 24-well plate were infected with C. jejuni (B) 11168H, (C) 81-176 or (D) 488 wild-type strains or treated with (E) thapsigargin for 6 or 24 h at 37°C in a 5% CO 2 incubator. Medium from each well was subjected to human IL-8 ELISA to measure the concentrations of IL-8. One sample t -test was performed. Asterisks denote a statistically significant difference (* = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: qRT-PCR showing expression of human (A, C) CHOP and (B, D) spliced XBP1 [ XBP1(s) ] in T84 or Caco-2 cells infected with either C. jejuni 11168H, 81-176 or 488 wild-type strains (MOI of 200:1) or treated with 2 µM of thapsigargin for 24 h at 37°C in a 5% CO 2 atmosphere. Prior to C. jejuni infection or thapsigargin treatment, T84 and Caco-2 cells were treated with 30 µM of ML130 for 4 h at 37°C in a 5% CO 2 incubator. T84 and Caco-2 cells were further treated with 30 µM of ML130 during C. jejuni infection or thapsigargin treatment. GAPDH was used as an internal control. (E) T84 cells were pre-treated with 30 µM of ML130 for 4 h at 37°C in a 5% CO 2 incubator. T84 cells were then infected with C. jejuni 11168H, 81-176 or 488 wild-type strains (MOI of 200:1) or treated with 2 µM thapsigargin for 24 h at 37°C in a 5% CO 2 incubator. T84 cells were further treated with 30 µM of ML130 for 4 h during C. jejuni infection. Medium from each well was subjected to human IL-8 ELISA to measure the concentrations of IL-8. One sample t -test was performed. Asterisks denote a statistically significant difference (* = p < 0.05; ** = p < 0.01; **** = p < 0.0001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Quantitative RT-PCR, Expressing, Infection, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: T84 cells grown in a 96-well plate were pre-treated with 5, 10, 20 or 30 µM of BAPTA-AM for 1 h at 37°C in a 5% CO 2 incubator. T84 cells were then infected with C. jejuni 11168H, 81-176 or 488 wild-type strains (MOI of 200:1) or treated with 2 µM thapsigargin for 24 h at 37°C in a 5% CO 2 incubator. After infection or treatment, 5 μM of cell-permeable dye Fura-2, AM was used to detect Ca 2+ . The two sets of fluorescence with 340 nm excitation and 510 nm emission (λ 1 ) and 380 nm excitation and 510 nm emission (λ 2 ) were recorded and intracellular Ca 2+ concentration was calculated . qRT-PCR showing expression of human (B) CHOP and (C) spliced XBP1 [ XBP1(s) ] in T84 cells infected with either C. jejuni 11168H wild-type strain (MOI of 200:1) or treated with 2 µM of thapsigargin for 24 h at 37°C in a 5% CO 2 atmosphere. Prior to C. jejuni infection or thapsigargin treatment, T84 cells were pre-treated with 10 µM of BAPTA-AM for 1 h at 37°C in a 5% CO 2 incubator. GAPDH was used as an internal control. One sample t -test was performed. Asterisks denote a statistically significant difference (** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Infection, Fluorescence, Concentration Assay, Quantitative RT-PCR, Expressing, Control

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: qRT-PCR showing expression of human (A) CHOP and (B) spliced XBP1 [ XBP1(s) ] in T84 cells infected with either C. jejuni 11168H wild-type strain, mutants or heat-killed 11168H wild-type strain for 24 h at 37°C in a 5% CO 2 atmosphere (MOI of 200:1). (C) RT-PCR showing expression of unspliced and spliced XBP1 [ XBP1(u) and XBP1(s) ] in T84 and Caco-2 cells infected with either C. jejuni 11168H wild-type strain or cdtABC operon mutant for 24 h at 37°C in a 5% CO 2 atmosphere. GAPDH was used as an internal control. One sample t -test was performed. Asterisks denote a statistically significant difference (** = p < 0.01; *** = p < 0.001; **** = p < 0.0001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Quantitative RT-PCR, Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Control

Journal: bioRxiv

Article Title: Unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence

doi: 10.1101/2025.01.31.635839

Figure Lengend Snippet: Interactions with and invasion of T84 intestinal epithelial cells by C. jejuni 11168H wild-type strain and different mutants . T84 cells were infected with C. jejuni 11168H wild-type strain, 11168H mutants or heat-killed 11168H wild-type strain for 3 h at 37°C in a 5% CO 2 incubator (MOI of 200:1). (A) For interaction assays, T84 cells were then washed with PBS and lysed with 0.1% (v/v) Triton X-100 and CFU/ml were recorded after incubation. (B) For invasion assays, T84 cells were then incubated with gentamicin (150 µg/ml) for 2 h to kill extracellular bacteria and then lysed with 0.1% (v/v) Triton X-100 and CFU/ml were recorded after incubation. Three biological and three technical replicates were performed for each experiment. # denotes no growth was observed. Asterisks denote a statistically significant difference (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Article Snippet: Human carcinoma cell line T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Infection, Incubation, Bacteria

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