Journal: Journal of Pediatric Gastroenterology & Nutrition

Article Title: Human Milk Oligosaccharides Protect Against Enteropathogenic Escherichia coli Attachment In Vitro and EPEC Colonization in Suckling Mice

doi: 10.1097/mpg.0000000000000172

Figure Lengend Snippet: FIGURE 1. Human milk oligosaccharides (HMOs) block adherence of enteropathogenic Escherichia coli (EPEC) in vitro. A, Cultures of 80% to 90% confluent HeLa, T84, or HEp-2 cells in 6-well plates were infected with EPEC (3 106 CFU) previously incubated with HMO (10 mg/mL) or medium for 1 hour, then bacterial attachment (% of inoculum) was assessed; data are shown as mean standard error of the mean (SEM). Indication of significant difference by Mann- Whitney test compared with medium alone, P 0.01, P 0.0001. B, HeLa cells were infected with EPEC (3 106) for 1 hour previously incubated with different amounts of HMO or galactooligosaccharides (control) for indicated time (1 or 2 hours), bacterial attachment was assessed thereafter. Data are shown as mean SEM, indication of significant difference by 1-sample t test compared with control, P 0.01, P 0.0001. C, HeLa cells were preincubated with HMOs (10 mg/mL) for 1 hour, washed, EPEC was added for 1 hour, and then bacterial attachment was assessed; data are shown as mean SEM compared with initial inoculum (3 106). All cell culture experiments were at least performed 3 times.

Article Snippet: The human colon epithelial cell line T84 (ATCC CCL-248) was seeded onto 42-mm2 permeable filter supports (EMD Millipore, Billerica, MA) in 6-well plates and grown to confluence for 10 to 14 days to reach a minimum transepithelial resistance of 1000/Vcm2.

Techniques: Blocking Assay, In Vitro, Infection, Incubation, MANN-WHITNEY, 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: 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: PLoS ONE

Article Title: Campylobacter jejuni Type VI Secretion System: Roles in Adaptation to Deoxycholic Acid, Host Cell Adherence, Invasion, and In Vivo Colonization

doi: 10.1371/journal.pone.0042842

Figure Lengend Snippet: (A) T84 adhesion efficacy, B) T84 invasion efficacy, (C) RAW 267.4 macrophage adhesion efficacy and (D) RAW 267.4 macrophage invasion efficacy. The graph represents results from three independent experiments. Error bars represent the standard error of the mean. P value: *≤0.05, **≤0.01, ***≤0.001.

Article Snippet: A stock culture of T84 (a human colonic epithelial cell line; ATCC, CCL 6) was maintained in 1∶1 mixture of Han's F12 and Dulbecco's modified Eagles medium (DMEM∶F12; ATCC) supplemented with 5% (vol/vol) fetal bovine serum, penicillin (100 U ml −1 ) and streptomycin (100 μg ml −1 ) according to ATCC's recommendation.

Techniques:

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

Journal: Journal of Biological Chemistry

Article Title: Aeromonas hydrophila Cytotoxic Enterotoxin Activates Mitogen-activated Protein Kinases and Induces Apoptosis in Murine Macrophages and Human Intestinal Epithelial Cells

doi: 10.1074/jbc.m404641200

Figure Lengend Snippet: FIG. 8. Demonstration of Act-induced blebbing of RAW 264.7 cells and T84 cells by laser scanning confocal microscopy and demonstration of apoptosis-associated cell morphology in RAW 264.7 cells by electron microscopy. Cells were grown on covered glass slides, 20 ng/ml Act was added, and alteration of cell morphology was observed by confocal microscopy. For electron microscopy, RAW 264.7 cells were treated with 20 ng/ml Act for 3 h, the cells were fixed, embedded, and stained before observation. Panel A, confocal micrographs of untreated RAW 264.7 cells. Panel B, confocal micrographs of RAW 264.7 cells treated with Act for 2.5 h. Panel C, confocal micrographs of RAW 264.7 cells treated with Act for 4 h. Black arrows indicate membrane blebbing of individual cells undergoing apoptosis. Panel D, electron micrograph of Act-treated RAW 264.7 cells. Note cellular density and condensation and margination of chromatin, and blebbing of the cytoplasm fragments off the cell. Panel E, confocal micrographs of untreated T84 cells. Panel F, confocal micrographs of T84 cells treated with Act for 5 h. Black arrows indicate membrane blebbing. Act-induced apoptosis of intestinal epithelial cells was confirmed by incubation of Act-treated HT-29 cells with FITC-conjugated annexin V and propidium iodide and then examination by flow cytometry. Panel G, untreated HT-29 cells were used as a negative control. Panel H, HT-29 cells were treated with 25 ng/ml Act for 6 h. Panel I, cycloheximide (100 g/ml)-treated HT-29 cells served as a positive control for apoptosis. Quadrant 2 represents late-apoptotic or necrotic cells, quadrant 3 represents viable cells, and quadrant 4 represents early apoptotic cells. Only 4.9% of the untreated cells were apoptotic (negative control), whereas 23.5% of the cycloheximide-treated cells were apoptotic (positive control). By 6 h, Act induced 34.0% apoptosis of HT-29 cells (H).

Article Snippet: Cell Culture—The murine macrophage (RAW 264.7) and human intestinal epithelial (T84 and HT-29) cell lines were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Confocal Microscopy, Electron Microscopy, Staining, Membrane, Incubation, Flow Cytometry, Negative Control, Positive Control