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: The Journal of Biological Chemistry

Article Title: Spermidine Stimulates T Cell Protein-tyrosine Phosphatase-mediated Protection of Intestinal Epithelial Barrier Function *

doi: 10.1074/jbc.M113.475962

Figure Lengend Snippet: Spermidine increases TCPTP expression at the protein level in T84 and HT29 intestinal epithelial cells. A, SPD (0.1, 1.0, and 10 μm) increased TCPTP protein levels in T84 (A) and HT29 cells (B) in a dose-dependent manner following 24 h treatment (n = 4). Unt, untreated. C, SPD (0.1, 1.0, 10 μm) showed no effect on PTPN2 mRNA expression in T84 cells following 24-h treatment (n = 6). PTPN2 mRNA expression was normalized to the housekeeping gene GAPDH. D, cycloheximide (CHX, 35.5 μm) significantly reduced TCPTP protein levels following 24 h treatment. Coadministration of SPD (10 μm) reversed this effect (n = 4). Whole cell lysates from SPD and CHX-treated cells were obtained, and TCPTP protein levels were assessed by Western blot analysis. β-Actin was used throughout as a loading control, and the relative protein levels were assessed by densitometry. All data are expressed as a percentage of the control ± S.E. *, p < 0.05 compared with untreated control; ###, p < 0.001 compared with CHX-treated cells; Student's unpaired t test or ANOVA and Student-Newman-Keuls post hoc test.

Article Snippet: Tissue Culture Human T 84 colonic epithelial cells (passages 20–40) were grown in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium with l -glutamine and 15 m m HEPES (Mediatech Inc., Manassas, VA), supplemented with 5% newborn calf serum (Invitrogen) and 1% penicillin/streptomycin (Cellgro/Mediatech, Herndon, VA) at 37 °C in 5% CO 2 .

Techniques: Expressing, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Spermidine Stimulates T Cell Protein-tyrosine Phosphatase-mediated Protection of Intestinal Epithelial Barrier Function *

doi: 10.1074/jbc.M113.475962

Figure Lengend Snippet: Spermidine increases TCPTP enzymatic activity in T84 and HT29 intestinal epithelial cells. T84 (A) and HT29 (B) cells were treated with SPD (10 μm) for 30 min. TCPTP was immunoprecipitated from whole cell lysates, and TCPTP activity was assessed (n = 3). A sample from each immunoprecipitation was probed for TCPTP by Western blotting to confirm equal protein loading. Fluorescence activity units were compared with TCPTP protein levels to account for any differences in overall phosphatase amounts. All data are expressed as a percentage of the control ± S.E. *, p < 0.05; **, p < 0.01 compared with the respective untreated time point; Student's unpaired t test.

Article Snippet: Tissue Culture Human T 84 colonic epithelial cells (passages 20–40) were grown in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium with l -glutamine and 15 m m HEPES (Mediatech Inc., Manassas, VA), supplemented with 5% newborn calf serum (Invitrogen) and 1% penicillin/streptomycin (Cellgro/Mediatech, Herndon, VA) at 37 °C in 5% CO 2 .

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Fluorescence, Control

Journal: The Journal of Biological Chemistry

Article Title: Spermidine Stimulates T Cell Protein-tyrosine Phosphatase-mediated Protection of Intestinal Epithelial Barrier Function *

doi: 10.1074/jbc.M113.475962

Figure Lengend Snippet: Spermidine attenuates STAT1 and 3 phosphorylation in IFN-γ-treated T84 and HT29 intestinal epithelial cells. IFN-γ (1000 units/ml) induced phosphorylation of STAT1 and 3 in T84 (A and B) and HT29 (C and D) cells following 30-min treatment. Coadministration of SPD (10 μm) for this time significantly attenuated IFN-γ-induced phosphorylation of STAT1 and 3 in both T84 and HT29 cells (n = 3–5). Whole cell lysates from IFN-γ and/or SPD-treated T84 and HT29 cells were obtained, and STAT1 and 3 protein levels were assessed by Western blot analysis. β-Actin was used throughout as a loading control, and the relative protein levels were assessed by densitometry. All data are expressed as a percentage of the control ± S.E. **, p < 0.01; ***, p < 0.001 compared with untreated control; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with IFN-γ-treated cells; ANOVA and Student-Newman-Keuls post hoc test. Unt, untreated.

Article Snippet: Tissue Culture Human T 84 colonic epithelial cells (passages 20–40) were grown in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium with l -glutamine and 15 m m HEPES (Mediatech Inc., Manassas, VA), supplemented with 5% newborn calf serum (Invitrogen) and 1% penicillin/streptomycin (Cellgro/Mediatech, Herndon, VA) at 37 °C in 5% CO 2 .

Techniques: Phospho-proteomics, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Spermidine Stimulates T Cell Protein-tyrosine Phosphatase-mediated Protection of Intestinal Epithelial Barrier Function *

doi: 10.1074/jbc.M113.475962

Figure Lengend Snippet: Spermidine inhibits PI3K activation by IFN-γ and protects intestinal epithelial barrier function from inflammatory cytokine treatment. A, T84 cells (1 × 105) were seeded onto coverslips and allowed to grow for 2 days. On day 3, fresh medium (DMEM) was added, and cells were pretreated ± SPD (10 μm) for 15 min. After 15 min, the cells were treated with IFN-γ (1000 units/ml) for 15 min. PI3K activation was detected with a rabbit anti-phospho-PI3K antibody (phospho-p85Tyr-458/p55Tyr-199; 1:50 dilution; green). DNA was stained with DAPI (blue). IFN-γ treatment increased PI3K phosphorylation, and this was reduced in the presence of SPD (representative image from five fields of view, ×20, n = 2, scale bar = 200 μm). B, IFN-γ (1000 units/ml) was added basolaterally to T84 monolayers that had stable TER values of ≥ 1000 Ω/cm2. Following 24-h treatment, TER values decreased by 35% of their initial base-line measurements. Coadministration of SPD (10 μm) for this time significantly reduced the effects of IFN-γ on barrier resistance (n = 4). C, following 24-h treatment, IFN-γ (1000 units/ml) also induced increases in epithelial permeability as measured by FITC-dextran flux. Coadministration of SPD (10 μm) for this time significantly reduced the effects of IFN-γ on barrier permeability (n = 4). Data are expressed as a percentage of the untreated control ± S.E. *, p < 0.05; ***, p < 0.001 compared with untreated cells; #, p < 0.05 compared with IFN-γ-treated cells; ANOVA and Student-Newman-Keuls post hoc test. UNT, untreated.

Article Snippet: Tissue Culture Human T 84 colonic epithelial cells (passages 20–40) were grown in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium with l -glutamine and 15 m m HEPES (Mediatech Inc., Manassas, VA), supplemented with 5% newborn calf serum (Invitrogen) and 1% penicillin/streptomycin (Cellgro/Mediatech, Herndon, VA) at 37 °C in 5% CO 2 .

Techniques: Activation Assay, Staining, Phospho-proteomics, Permeability, Control

Journal: The Journal of Biological Chemistry

Article Title: Spermidine Stimulates T Cell Protein-tyrosine Phosphatase-mediated Protection of Intestinal Epithelial Barrier Function *

doi: 10.1074/jbc.M113.475962

Figure Lengend Snippet: Spermidine protects intestinal epithelial barrier function in a TCPTP-dependent manner. T84 cells were transfected with either TCPTP-specific or nonspecific control siRNA and allowed to grow for 48 h, at which point IFN-γ (1000 units/ml) was added basolaterally to the monolayers. Following 24 h treatment, TER values decreased on average by 8% of their initial base-line measurements. Coadministration of SPD (10 μm) for this time significantly reduced the effects of IFN-γ on barrier resistance (n = 5). The protective effect of SPD on IFN-γ-induced decreases in TER was not realized in TCPTP knockdown cells (n = 5). Whole cell lysates from IFN-γ- and/or SPD-treated T84 cells were obtained, and TCPTP protein levels were assessed by Western blot analysis. **, p < 0.01; ***, p < 0.001 compared with untreated cells; #, p < 0.05 compared with IFN-γ treatment of control siRNA-transfected cells; $$, p < 0.01 compared with IFN-γ/SPD treatment of control siRNA-transfected cells. Unt, untreated.

Article Snippet: Tissue Culture Human T 84 colonic epithelial cells (passages 20–40) were grown in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium with l -glutamine and 15 m m HEPES (Mediatech Inc., Manassas, VA), supplemented with 5% newborn calf serum (Invitrogen) and 1% penicillin/streptomycin (Cellgro/Mediatech, Herndon, VA) at 37 °C in 5% CO 2 .

Techniques: Transfection, Control, Knockdown, Western Blot

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