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:

Article Title: Airway epithelial cells synthesize the lipid mediator 5-oxo-ETE in response to oxidative stress

doi: 10.1016/j.freeradbiomed.2006.12.006

Figure Lengend Snippet: Epithelial cells used in this study

Article Snippet: Epithelial cells lines (A549, BEAS-2B, T84, and HEp-2) were obtained from the American Type Culture Collection ( ) and cultured in 10% FBS in DMEM/F12 medium containing L-glutamine (2 mM), sodium bicarbonate (2.5 g/L), penicillin (100U/ml), and streptomycin (100 μg/ml).

Techniques:

Journal:

Article Title: Airway epithelial cells synthesize the lipid mediator 5-oxo-ETE in response to oxidative stress

doi: 10.1016/j.freeradbiomed.2006.12.006

Figure Lengend Snippet: Epithelial cells were cultured to confluence in 6-well plates. Cells were washed twice with PBS+ and then preincubated for 6 min with 100 μM PMS. The cells were then incubated for an additional 20 min with 4 μM 5-HETE. Cells in adjoining wells were detached with trypsin and counted. Values are means ± SE (n = 4, except for T84 and HEp-2 cells (n=2))..

Article Snippet: Epithelial cells lines (A549, BEAS-2B, T84, and HEp-2) were obtained from the American Type Culture Collection ( ) and cultured in 10% FBS in DMEM/F12 medium containing L-glutamine (2 mM), sodium bicarbonate (2.5 g/L), penicillin (100U/ml), and streptomycin (100 μg/ml).

Techniques: Cell Culture, Incubation

Journal:

Article Title: Airway epithelial cells synthesize the lipid mediator 5-oxo-ETE in response to oxidative stress

doi: 10.1016/j.freeradbiomed.2006.12.006

Figure Lengend Snippet: Scheme showing the regulation of 5-oxo-ETE synthesis in airway epithelial cells. Metabolism of H2O2 and tBuOOH by glutathione peroxidase (GPerox) results in the formation of GSSG, which is reduced back to GSH by glutathione reductase (GRed), generating NADP+, which serves as a cofactor for 5-HEDH. This process can be blocked by NEM, which alkylates GSH, and mimicked by diamide (DA), which oxidizes GSH, and PMS, which oxidizes NADPH. In A549 cells the action of H2O2 is limited by metabolism by catalase, which can be blocked by azide.

Article Snippet: Epithelial cells lines (A549, BEAS-2B, T84, and HEp-2) were obtained from the American Type Culture Collection ( ) and cultured in 10% FBS in DMEM/F12 medium containing L-glutamine (2 mM), sodium bicarbonate (2.5 g/L), penicillin (100U/ml), and streptomycin (100 μg/ml).

Techniques:

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: 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: Cancers

Article Title: Oncogenic Signalling of PEAK2 Pseudokinase in Colon Cancer

doi: 10.3390/cancers14122981

Figure Lengend Snippet: PEAK2 expression and tyrosine phosphorylation in CRC. ( A , B ): PEAK2 protein levels and Y413 phosphorylation in established CRC cell lines ( A ) and patient-derived CRC lines ( B ). Left panel: Western blotting showing the levels of PEAK2, pY413 PEAK2, SRC, and pY418 SRC from indicated CRC cell lines and CRC patient-derived lines. Right: correlation between the mean ( n = 3) of relative pY413 PEAK2 level (fold control) and relative SKF activity (pY418 SRC level, fold control) from indicated CRC or CTC cells. ( C ) Patients with CRC showing high PRAG1 expression have shorter free recurrence survival (FRS). Kaplan–Meier analysis using data from 205 patients with stage III CRC subdivided according to the tumour PRAG1 expression level (high/low) ( p -value: 0.0450).

Article Snippet: CRC cell lines (HCT116, HT29, Caco2, SW480, SW620, Lovo, LS174T, DLD1, T84, and Colo205) and HEK293T cell lines were obtained from ATCC (Rockville, MD, USA); C0-115 (a gift from Dr Pierre Roux, CNRS of Montpellier) was described in [ ].

Techniques: Expressing, Phospho-proteomics, Derivative Assay, Western Blot, Control, Activity Assay

Journal: Cancers

Article Title: Oncogenic Signalling of PEAK2 Pseudokinase in Colon Cancer

doi: 10.3390/cancers14122981

Figure Lengend Snippet: PEAK2 regulates CRC cell morphology and invasion. PEAK2 depletion by shRNA inhibits CRC cell growth and invasion. ( A ) Cellular protein tyrosine phosphorylation and PEAK2 level in CRC cells transduced with indicated shRNA (left: a representative example, right: quantification). ( B ) Morphology of CRC cells transduced with indicated shRNA (left: a representative example, right: quantification of the % of elongated cells). ( C ) Invasion of CRC cells transduced with indicated shRNA in Boyden chambers coated with matrigel (left: presentative image, right: quantification of number of invaded cells). Mean ± SEM; n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t -test).

Article Snippet: CRC cell lines (HCT116, HT29, Caco2, SW480, SW620, Lovo, LS174T, DLD1, T84, and Colo205) and HEK293T cell lines were obtained from ATCC (Rockville, MD, USA); C0-115 (a gift from Dr Pierre Roux, CNRS of Montpellier) was described in [ ].

Techniques: shRNA, Phospho-proteomics, Transduction

Journal: Cancers

Article Title: Oncogenic Signalling of PEAK2 Pseudokinase in Colon Cancer

doi: 10.3390/cancers14122981

Figure Lengend Snippet: PEAK2 depletion reduces CRC cell growth and tumour development. ( A ) Anchorage-independent growth in soft agar of CRC cells expressing shRNA control (shCtrl) or shRNA PEAK2 (shPEAK2) (left: representative example; right: quantification of colonies obtained in soft-agar. ( B , C ) Tumour development in nude mice subcutaneously inoculated with indicated CRC cells that were transduced with shRNA control (shCtrl) or shRNA PEAK2 (shPEAK2). Is shown a representative example of tumours obtained in nude mice, the time-course of tumour development (volume) and the tumour mass; mean ± SEM from 13 ( B ) and 17 ( C ) mice per condition (* p < 0.05; ** p < 0.01; *** p < 0.001) (Student’s t -test).

Article Snippet: CRC cell lines (HCT116, HT29, Caco2, SW480, SW620, Lovo, LS174T, DLD1, T84, and Colo205) and HEK293T cell lines were obtained from ATCC (Rockville, MD, USA); C0-115 (a gift from Dr Pierre Roux, CNRS of Montpellier) was described in [ ].

Techniques: Expressing, shRNA, Control, Transduction

Journal: Cancers

Article Title: Oncogenic Signalling of PEAK2 Pseudokinase in Colon Cancer

doi: 10.3390/cancers14122981

Figure Lengend Snippet: PEAK2 expression promotes CRC cell growth, adhesion and invasion. ( A ) PEAK2 expression increases growth of HCT116 CRC cells. Is shown the cellular protein tyrosine phosphorylation, PEAK2 protein levels and anchorage independent growth of HCT116 cells transduced with indicated PEAK2 constructs. The quantification of protein tyrosine phosphorylation and colonies formation in soft agar (% control) is shown from 3 independent experiments. ( B ) SRC expression promotes PEAK2 oncogenic function in SW620 CRC cells. Protein tyrosine phosphorylation, PEAK2 protein level and phosphorylation on Y413 and colonies formation in soft-agar of SW620 cells that were coinfected with SRC, PEAK2 Myc or control viruses (mock, PMX) as shown. The relative quantification of pY413 PEAK2 levels and colonies formation is shown from 3 independent experiments. ( C ) Adhesion on fibronectin and invasion in matrigel of indicated SW620 cells. Is shown the mean ± SEM; n = 3–4 * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t -test).

Article Snippet: CRC cell lines (HCT116, HT29, Caco2, SW480, SW620, Lovo, LS174T, DLD1, T84, and Colo205) and HEK293T cell lines were obtained from ATCC (Rockville, MD, USA); C0-115 (a gift from Dr Pierre Roux, CNRS of Montpellier) was described in [ ].

Techniques: Expressing, Phospho-proteomics, Transduction, Construct, Control, Quantitative Proteomics

Journal: Cancers

Article Title: Oncogenic Signalling of PEAK2 Pseudokinase in Colon Cancer

doi: 10.3390/cancers14122981

Figure Lengend Snippet: PEAK2 induces ABL-dependent folipodia in CRC cells. ( A ) PEAK2 colocalizes with F-actin structures at focal adhesion of CRC cells. Immunostaining of PEAK2 and F-actin in SW480 cells expressing indicated PEAK2 Myc constructs that were seeded on fibronectin. ( B , C ) PEAK2 induces ABL-dependent filopodia. A representative example ( B ) and quantification ( C ) of filopodia in HCT116 transfected with indicated PEAK2 Myc constructs and treated with DMSO (0.1%) or ABLi (100 nM nilotinib) as indicated for 2 h before cell fixation; mean ± SEM; n = 3; ** p < 0.01; *** p < 0.001 Student’s t -test).

Article Snippet: CRC cell lines (HCT116, HT29, Caco2, SW480, SW620, Lovo, LS174T, DLD1, T84, and Colo205) and HEK293T cell lines were obtained from ATCC (Rockville, MD, USA); C0-115 (a gift from Dr Pierre Roux, CNRS of Montpellier) was described in [ ].

Techniques: Immunostaining, Expressing, Construct, Transfection

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