Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Gene expression analysis of Toll-like receptors (TLR2, TLR5, TLR7) ( a ), E-cadherin and ZO-1 ( b ), claudins (CLDN1–4) ( d ), and the brush-border enzyme alkaline phosphatase (ALP) ( c ) in Caco-2 and T84 cells at day 0, 7, 14, and 21. All experiments were performed as three independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of three independent experiments. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared to Caco-2 Day 0. Error bars represent the standard error of the mean (SEM) from two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Gene Expression

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Transepithelial electrical resistance (TEER) of Caco-2 and T84 monolayers cultured in Transwell ® inserts and maintained in respective media. All experiments were performed as three independent biological replicates. The data presented are from one representative experiment out of three independent experiments. Error bars represent the standard error of the mean (SEM) of replicates in one experiment.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Culture

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Identification and localization of ZO-1 in Caco-2 ( a ) and T84 ( b ) cells by immunofluorescence staining of ZO-1 (Alexa Fluor™ 488, green) at Day 0, Day 7, Day 14, and Day 21 after seeding. Nuclei are counterstained with DAPI (blue), and fluorescence channels were overlaid using FIJI (ImageJ, version 1.54g). Images are representative of three independent experiments.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Effect of peanut allergens on cell proliferation of Caco-2 ( a ) and T84 ( b ) monolayers after 4 h stimulation with 1 mg/mL peanut protein extracts (Raw and Roasted). All experiments were performed as three independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of three independent experiments. Statistical significance is denoted as ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared to untreated cells (Only Cells). Error bars represent the standard error of the mean (SEM) of two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Effect of peanut proteins on barrier integrity of Caco-2 ( a ) and T84 ( b ) monolayers after 4 h stimulation with 1 mg/mL peanut protein extracts (Raw and Roasted). All experiments were performed as three independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of three independent experiments. Statistical significance is indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared to untreated cells (Only Cells). Error bars represent the standard error of the mean (SEM) of two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Western blot detection of Ara h 1 in samples collected from basolateral compartments of Caco-2 and T84 cell monolayers treated with 1 mg/mL of raw or roasted peanut extracts for 4 h. A purified Ara h 1 standard (10 µg) was included as a positive control and molecular reference. Samples were analyzed by Western blot using rabbit polyclonal anti-Ara h 1 antibodies.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Purification, Positive Control

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Passage of Ara h 1 across intestinal epithelial barriers in Caco-2 and T84 cell monolayers following 4 h exposure to 1 mg/mL peanut extract (Raw and Roasted), shown as percentage inhibition ( a ) and percentage transport efficiency ( b ). Transport studies were performed on supernatants collected from three independent biological experiments and the data presented are from one representative experiment out of three independent experiments. Statistical significance is indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with untreated control cells (Only Cells). Error bars represent the standard error of the mean (SEM) from two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Inhibition, Control

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Assessment of peanut allergen effects on tight junction protein ZO-1 in both 14 day differentiated Caco-2 ( a ) and T84 ( b ) monolayers after 4 h treatment with 1 mg/mL peanut protein extracts (Raw and Roasted). Untreated cells (OC) are used as a negative control. All groups are subsequently stained for ZO-1 (Alexa Fluor™ 488, green). Nuclei are counterstained with DAPI (blue), and fluorescence channels were overlaid using FIJI (ImageJ). Images are representative of three independent experiments.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Negative Control, Staining, Fluorescence

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Transcriptional analysis of Cytokines IL1β ( a ) TSLP ( b ) IL25 ( c ), and IL33 ( d ) released by Caco-2 and T84 epithelial cells after 4 h and 24 h exposure to raw and roasted peanut protein extracts (1 mg/mL), purified Ara h 1 (raw or roasted, 0.1 mg/mL), LPS (1 µg/mL) and gelatin (1 mg/mL). Untreated cells (Only Cells) are used as a negative control. All experiments were performed as two independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of two independent experiments. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with untreated control cells (Only Cells). Error bars represent the standard error of the mean (SEM) from two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Purification, Negative Control, Control

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Detection of Cytokines IL-1β ( a ) TSLP ( b ) IL-25 ( c ), and IL-33 ( d ) released by Caco-2 and T84 epithelial cells after 4 h and 24 h exposure to raw and roasted peanut protein extracts (1 mg/mL), purified Ara h 1 (raw or roasted, 0.1 mg/mL), LPS (1 µg/mL) and gelatin (1 mg/mL). Untreated cells (Only Cells) are used as a negative control. All experiments were performed in two independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of two independent experiments. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with untreated cells (Only Cells). Error bars represent the standard error of the mean (SEM) from two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Purification, Negative Control

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Measurement of ROS production at 4 and 24 h in Caco-2 ( a ) and T84 ( b ) cells after treatment with raw or roasted peanut protein extracts (1 mg/mL), purified Ara h 1 (raw or roasted, 0.1 mg/mL), LPS (1 µg/mL) and gelatin (1 mg/mL). Untreated cells (Only Cells) are used as negative control. All experiments were performed in two independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of two independent experiments. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with untreated control cells (Only Cells). Error bars represent the standard error of the mean (SEM) from two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Purification, Negative Control, Control

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: Measurement of NO production at 4 and 24 h in Caco-2 ( a ) and T84 ( b ) cells after treatment with raw or roasted peanut protein extracts (1 mg/mL), purified Ara h 1 (raw or roasted, 0.1 mg/mL), LPS (1 µg/mL) and gelatin (1 mg/mL). Untreated cells (Only Cells) were used as a negative control. All experiments were performed in two independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of two independent experiments. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with untreated control cells (Only Cells). Error bars represent the standard error of the mean (SEM) from two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Purification, Negative Control, Control

Journal: Foods

Article Title: Modeling Early Events in Food Sensitization: Complementary Insights from Caco-2 and T84 Epithelial Barriers Exposed to Peanut Allergens

doi: 10.3390/foods15050825

Figure Lengend Snippet: IL-6 secretion from THP-1-derived macrophages. THP-1-derived macrophages were stimulated with basolateral supernatants collected following 4 h exposure of differentiated Caco-2 and T84 epithelial monolayers to raw or roasted peanut protein extracts (1 mg/mL). ( a ) A schematic illustration generated using BioRender ( https://BioRender.com/ ; accessed on 19 January 2026) depicts the experimental workflow, in which basolateral media containing translocated allergens were collected after epithelial exposure and subsequently applied to THP-1-derived macrophages. ( b ) IL-6 secretion was quantified using a sandwich ELISA. Untreated cells (Only Cells) served as a negative control. All experiments were performed as three independent biological replicates, with each experimental group analyzed in duplicate. The data presented are from one representative experiment out of three independent experiments. Statistical significance is denoted as * p < 0.05, ** p < 0.01, compared with untreated control cells (Only Cells). Error bars represent the standard error of the mean (SEM) of two replicates/group.

Article Snippet: Caco-2, T84, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Derivative Assay, Generated, Sandwich ELISA, Negative Control, Control

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: Scientific Reports

Article Title: CEACAM1 mediates B cell aggregation in central nervous system autoimmunity

doi: 10.1038/srep29847

Figure Lengend Snippet: B cell aggregation was induced by 16 h of incubation with anti-CD19 ( A , compare untreated to anti-CD19-treated cells). ( B – D ) Inhibition of CD19-induced B cell aggregation by the anti-CEACAM1 antibody clones C5-1X (150 μg/ml) ( B ), 4D1/C2 (150 μg/ml) ( C ) and T84.1 (150, 250 vs . 400 μg/ml) ( D ). B cells were isolated from n = 3 ( B,C ) or n = 9 ( D ) healthy donors, respectively. ( E ) Induction of B cell aggreation by anti-CD19 antibody and its inhibition by T84.1 in age- and gender-matched MS patients ( n = 4) and healthy controls ( n = 4).

Article Snippet: The three clones C5-1X (Reliatech), 4D1/C2 (produced and purified in-house) and T84.1 (inVivo BioTech Services) were tested at different concentrations (150 μg/ml, 250 μg/ml and 400 μg/ml).

Techniques: Incubation, Inhibition, Clone Assay, Isolation

Journal:

Article Title: Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells

doi: 10.1042/BJ20040021

Figure Lengend Snippet: T84 monolayers grown in DMEM supplemented with 10% FBS were serum starved for 24 h in 0.5% FBS. Adenosine (Ado; 100 μM) was then added to the apical (Ap) or basolateral (Bl) compartment. After an incubation period of 24 h, culture media in the apical or basolateral compartments were collected and processed for Western blotting to determine FN secretion. Protein bands in the autoradiogram shown in (A) were quantified by scanning densitometry, and densities relative to vehicle-treated controls are shown in the histogram to illustrate the alterations in band intensities. The data are representive of three separate experiments. (B) Confocal imaging of adenosine-induced FN in intestinal epithelial monolayer. Serum starved T84 cells (0.5% FBS, 24 h) were stimulated with apical or basolateral adenosine (100 μM) for 24 h, after which they were fixed and stained with anti-FN antibody followed by FITC-conjugated secondary antibody and counterstained with rhodamine/phalloidin, as described in the Experimental section. Vertical sections were taken off the monolayers to define the top (0 μm) and the bottom of the monolayer (18–20 mm). Shown here are ‘en face’ (x–y) images taken at the level of apical (1.2 μm above the level of tight junction), mid- (6 μm below the level of tight junction) and basal (18–20 μm, at the level of the stress fibres) pole of the epithelial monolayer.

Article Snippet: To determine if adenosine modulates FN secretion and whether this modulation is polarized, confluent monolayers of intestinal epithelial T84 and IEC-6 cells, grown in tissue culture inserts (Corning, Acton, MA, U.S.A.), were stimulated via the apical or basal surface with adenosine (0–100 μM), and conditioned media were analysed by Western blot analysis to examine the steady-state level of FN protein.

Techniques: Incubation, Western Blot, Imaging, Staining

Journal:

Article Title: Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells

doi: 10.1042/BJ20040021

Figure Lengend Snippet: T84 monolayers were serum starved in 0.5% FBS. After 24 h, cells were stimulated with adenosine (apical or basolateral; Ado, 100 μM), culture media were collected at 8, 16, 24 and 48 h, and Western blotting was performed as described in the Experimental section. Autoradiographs were quantified by densitometric image scanning and are expressed in the histograms as fold-increase over vehicle-stimulated controls. Data represent the responses observed in two separate experiments.

Article Snippet: To determine if adenosine modulates FN secretion and whether this modulation is polarized, confluent monolayers of intestinal epithelial T84 and IEC-6 cells, grown in tissue culture inserts (Corning, Acton, MA, U.S.A.), were stimulated via the apical or basal surface with adenosine (0–100 μM), and conditioned media were analysed by Western blot analysis to examine the steady-state level of FN protein.

Techniques: Western Blot

Journal:

Article Title: Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells

doi: 10.1042/BJ20040021

Figure Lengend Snippet: T84 cells growing in monolayer were treated with increasing doses of 0.005, 0.01, 0.05 or 0.1 μM of adenosine (Ado), at either the apical (A) or basolateral (B) side, and culture media were collected after 24 h. Western blotting was performed as described in the Experimental section. Autoradiographs were quantified by densitometric image scanning and expressed in the histograms as fold-increase over vehicle-stimulated controls. Data presented are representative of the responses observed in three separate experiments.

Article Snippet: To determine if adenosine modulates FN secretion and whether this modulation is polarized, confluent monolayers of intestinal epithelial T84 and IEC-6 cells, grown in tissue culture inserts (Corning, Acton, MA, U.S.A.), were stimulated via the apical or basal surface with adenosine (0–100 μM), and conditioned media were analysed by Western blot analysis to examine the steady-state level of FN protein.

Techniques: Western Blot

Journal:

Article Title: Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells

doi: 10.1042/BJ20040021

Figure Lengend Snippet: T84 cells were pretreated with cycloheximide (50 μM) for 30 min before the addition of apical or basolateral adenosine (100 μM). Alloxazine (50 μM) was added along with apical or basolateral adenosine. After 24 h, conditioned media were collected and FN protein levels were analysed by Western blot analysis as described in the Experimental section. The blot is representative of two separate experiments done in duplicate.

Article Snippet: To determine if adenosine modulates FN secretion and whether this modulation is polarized, confluent monolayers of intestinal epithelial T84 and IEC-6 cells, grown in tissue culture inserts (Corning, Acton, MA, U.S.A.), were stimulated via the apical or basal surface with adenosine (0–100 μM), and conditioned media were analysed by Western blot analysis to examine the steady-state level of FN protein.

Techniques: Western Blot

Journal:

Article Title: Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells

doi: 10.1042/BJ20040021

Figure Lengend Snippet: Filter-grown T84 cell monolayers, were either stimulated with vehicle or adenosine (100 μM) for 24 h (A and B), gently washed and incubated for 1 h at 37 °C with S. typhimurium SL3201 added to the apical chamber, or were co-incubated with FN (12 μg/ml; apical) and S. typhimurium for 1 h at 37 °C (C and D). The number of adherent and invasive bacteria were evaluated by counting the CFU after plating them on MacConkey agar plates as described in the Experimental section. The data are representative of three independent experiments and are expressed as the means±S.D. for triplicate dilutions. *Significantly different compared with S. typhimurium alone (P<0.01).

Article Snippet: To determine if adenosine modulates FN secretion and whether this modulation is polarized, confluent monolayers of intestinal epithelial T84 and IEC-6 cells, grown in tissue culture inserts (Corning, Acton, MA, U.S.A.), were stimulated via the apical or basal surface with adenosine (0–100 μM), and conditioned media were analysed by Western blot analysis to examine the steady-state level of FN protein.

Techniques: Incubation

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.01.003

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

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

Techniques: Binding Assay, Protein Binding

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.01.003

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

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

Techniques: Western Blot, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.01.003

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

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

Techniques: Staining, Recombinant, Dot Blot, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.01.003

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

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

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

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2022.01.003

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

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

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