Journal: Frontiers in Immunology

Article Title: Ten-Eleven Translocation (TET) Enzymes Modulate the Activation of Dendritic Cells in Allergic Rhinitis

doi: 10.3389/fimmu.2019.02271

Figure Lengend Snippet: The relative expression of TET1, TET2, and TET3 in PBMCs and DCs and selectively knocked down the expression of TET1 with transfection of recombinant adenovirus. (A) The histogram shows TET1 expressed highest among TET family in human peripheral mDCs, and pDCs by flow cytometric analysis. (B) Comparing to TET2 and TET3, TET1 expressed highest in both PBMCs and moDCs. (C) Infection efficiency of recombinant adenovirus in moDCs. (D) Expression of TET1 in adenovirus-transfected moDCs was assessed using western blot. GAPDH was used as an internal control.

Article Snippet: On day 5, immature moDCs were transfected with adenovirus at 300 multiplicity of infection (MOI) in the presence of polybrene (5 μL/mL, Cyagen biosciences, Guangzhou, China).

Techniques: Expressing, Transfection, Recombinant, Infection, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Ten-Eleven Translocation (TET) Enzymes Modulate the Activation of Dendritic Cells in Allergic Rhinitis

doi: 10.3389/fimmu.2019.02271

Figure Lengend Snippet: TET1 knockdown effect on phenotypic activation of non-atopic and atopic moDCs. Immature non-atopic and atopic moDCs (day 5) were transfected by control-shRNA, TET1-shRNA2, or TET1-shRNA3 adenovirus. After another 48 h, the expression of costimulatory molecules, including CD 40, HLA-DR, CD86, and CD80, were evaluated by flow cytometry. (A) The histograms show the typical comparisons of every costimulatory molecule expression among Control-shRNA-treated moDCs (dark green), TET1-shRNA2 (dark blue), and TET1-shRNA3-treated moDCs (magenta) and their isotype controls (light green, light blue, and pink, respectively). (B–E) Statistical analyses were conducted with Friedman test and Dunn's multiple comparisons tests ( n = 6 HC and 6 AR) * p < 0.05.

Article Snippet: On day 5, immature moDCs were transfected with adenovirus at 300 multiplicity of infection (MOI) in the presence of polybrene (5 μL/mL, Cyagen biosciences, Guangzhou, China).

Techniques: Knockdown, Activation Assay, Transfection, Control, shRNA, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Ten-Eleven Translocation (TET) Enzymes Modulate the Activation of Dendritic Cells in Allergic Rhinitis

doi: 10.3389/fimmu.2019.02271

Figure Lengend Snippet: Effect of TET1-inhibited moDCs on the proliferation of CFSE-labeled T helper (Th) cells. Immature non-atopic and atopic moDCs (day 5) were transfected by control-shRNA, TET1-shRNA2, or TET1-shRNA3 adenovirus. On day 7, different treatments of moDCs were counted and co-cultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled autologous Th cells at a ratio of 1:10 (1 × 10 4 : 1 × 10 5 ) for another 72 h. The proliferation of CD4 + T cells measured by CFSE dilution. Statistical analyses were conducted with Friedman test and Dunn's multiple comparisons tests ( n = 6 HC and 6 AR).

Article Snippet: On day 5, immature moDCs were transfected with adenovirus at 300 multiplicity of infection (MOI) in the presence of polybrene (5 μL/mL, Cyagen biosciences, Guangzhou, China).

Techniques: Labeling, Transfection, Control, shRNA, Cell Culture

Journal: Frontiers in Immunology

Article Title: Ten-Eleven Translocation (TET) Enzymes Modulate the Activation of Dendritic Cells in Allergic Rhinitis

doi: 10.3389/fimmu.2019.02271

Figure Lengend Snippet: Effect of TET1-inhibited moDCs on the differentiation of regulatory T cells. Immature non-atopic and atopic moDCs (day 5) were transfected by control-shRNA, TET1-shRNA2, or TET1-shRNA3 adenovirus. On day 7, moDCs were co-cultured with autologous CD4 + T cells at a ratio of 1:10 (1 × 10 4 : 1 × 10 5 ) for another 72 h. The proportion of CD4 + FoxP3 + Tregs in CD4 + T cells (A) , and the proportion of Treg subsets, including CD4 + CD45RA + FoxP3 lo resting Treg cells (rTregs, shown as I Treg), CD4 + CD45RA − FoxP3 high activated Treg cells (aTregs, shown as II Treg) and CD4 + CD45RA − FoxP3 lo non-suppressive T cells (shown as III Treg), in CD4 + T cells (B) were evaluated. (C,D) Statistical analyses were conducted with Friedman test and Dunn's multiple comparisons tests ( n = 6 HC and 6 AR) * p < 0.05.

Article Snippet: On day 5, immature moDCs were transfected with adenovirus at 300 multiplicity of infection (MOI) in the presence of polybrene (5 μL/mL, Cyagen biosciences, Guangzhou, China).

Techniques: Transfection, Control, shRNA, Cell Culture

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 1 Co-immunoprecipitation of CtIP and AdE1A. (a) Lysates from 293 cells were immunoprecipitated with antibodies against Ad5E1A(M58) or CtBP1 (E12) as shown. Co-immunopre- cipitating CtIP was detected by western blotting. (b) Lysates from 911 and 293 cells were immunoprecipitated with two CtIP rabbit polyclonal antibodies. Immunoprecipitates were fractionated on ‘urea gels’ and co-immunoprecipitating Ad5E1A detected by western blotting. (c) Lysates from MCF7 cells infected (and mock infected) with Ad5wt were immunoprecipitated for Ad5E1A. Co- immunoprecipitating proteins were fractionated on SDS gels and CtIP identified by western blotting. (d) Lysates from MCF7 cells infected with dl520 or dl1135 were immunoprecipitated with rabbit antibodies against CtIP. Co-immunoprecipitating proteins were fractionated on ‘urea gels’ in the absence of SDS and western blotted for Ad5E1A. (e) Lysates from Ad12E1-transformed human and rat cells were immunoprecipitated with an antibody against Ad12E1A. Co-immunoprecipitating proteins were fractionated on gels run in the presence of SDS and blotted for CtIP. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein; SDS, sodium dodecylsulphate.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Immunoprecipitation, Western Blot, Infection, Transformation Assay, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 2 CtIP binds directly to Ad5E1A. (i) [35S]methionine- labelled CtIP or (ii) [35S]methionine-labelled CtBP1 were incubated with GST-Ad5E1A proteins and polypeptides (25 mg) as shown by Coomassie Blue staining (iii). Bound CtIP or CtBP1 were fractioned by SDS–polyacrylamide gel electrophoresis and detected by fluorography and autoradiography. Input represents 5% of the amount added in the pull downs. AdE1A, adenovirus onco- protein early region 1A; CtBP1, C-terminal-binding protein; CtIP, C-terminal-binding protein interacting protein; GST, glutathione- S-transferase.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Incubation, Staining, Polyacrylamide Gel Electrophoresis, Autoradiography, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 4 Mapping the CtIP-binding site in Ad5E1A. (a) Dimen- sions of GST-Ad5E1A polypeptides used in the pull-down assays. (b) [35S]methionine-labelled CtIP was incubated with GST-Ad5E1A polypeptides (25 mg) as shown. A Coomassie Blue stained gel of the GST fragments is shown in the lower panel. (c) [35S]methionine CtIP, p107 and TBP were incubated with GST-Ad512SE1A, GST- Ad513SE1A and GST-Ad5CR3. Bound proteins were fractioned by SDS–polyacrylamide gel electrophoresis and visualized by fluorography and autoradiography. Densitometric scanning was used to quantify the proportion of each of the proteins bound. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein; GST, glutathione- S-transferase.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Binding Assay, Incubation, Staining, Polyacrylamide Gel Electrophoresis, Autoradiography

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 5 Analysis of the binding site for CtIP in the N-terminal region of Ad5E1A. (a) [35S]methionine-labelled CtIP was incubated with GST-Ad512SE1A proteins carrying the amino-acid substitutions shown (20 mg). Bound proteins were isolated with glutathione agarose, eluted, fractionated by SDS–PAGE and detected by fluorography and autoradiography. Binding to wt Ad512S and Ad513S E1A is also shown. Input represents 5% of the total added in the pull-downs. Coomassie Blue-stained gels of the GST-AdE1As are shown. (b) CtIP binding to E1A CR3 from different viral serotypes. [35S]methionine-labelled CtIP was incubated with GST fusion proteins containing the CR3 regions of AdE1As, from different viral serotypes (25 mg). Bound proteins were fractioned by SDS–PAGE and detected by fluorography and autoradiography. Input represents 10% of the total added. A Coomassie Blue-stained gel of the GST-CR3 proteins is shown in the lower panel. AdE1A, adenovirus oncoprotein early region 1A; CR, conserved region; CtIP, C-terminal-binding protein interacting protein; GST, glutathione-S-transferase; SDS–PAGE, sodium dodecylsulphate–polyacrylamide gel electrophoresis.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Binding Assay, Incubation, Isolation, SDS Page, Autoradiography, Staining, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 6 Complex formation of Ad5E1A, CtBP1 and CtIP. GST- Ad5E1A or GST-CtBP2 was incubated with [35S]-labelled CtIP in the presence of increasing concentrations of a peptide containing the PLDLS motif. Protein mixtures were incubated with glu- tathione agarose beads. The level of bound GST-Ad5E1A or GST- CtBP2 is shown by SDS–PAGE and staining for total protein (i). Bound [35S]-CtIP was detected by fluorography and autoradio- graphy (ii). AdE1A, adenovirus oncoprotein early region 1A; CtBP2, C-terminal-binding protein; CtIP, C-terminal-binding protein interacting protein; GST, glutathione-S-transferase.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Incubation, SDS Page, Staining, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 7 The effect of CtIP on Ad5E1A transactivation. CtIP or CtBP expression was reduced in A549 cells using appropriate siRNAs. Control cells were transfected with a nonspecific control siRNA. After 3 days, cells were transfected with pcDNA 3-Gal4 DBD and a Gal4-responsive luciferase reporter, pcDNA-Gal4 DBD-CR3 and a Gal4-responsive luciferase reporter or VP16 DBD and a Gal4-responsive luciferase reporter. (a) At 24 h post- transfection luciferase activity was determined. (b) Western blot showing reduction of CtIP expression (upper panel) and CtBP expression (lower panel) by siRNA. AdE1A, adenovirus oncopro- tein early region 1A; CtBP2, C-terminal-binding protein; CtIP, C- terminal-binding protein interacting protein; DBD, DNA-binding domain; GST, glutathione-S-transferase; siRNA, small interfering RNA.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Expressing, Control, Transfection, Luciferase, Activity Assay, Western Blot, Binding Assay, Small Interfering RNA

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 9 Ad5E1A disrupts complexes between CtIP and Rb and p130. (a) MCF7 cells were mock infected or infected with dl1520 (20 pfu/cell) for 24 h. CtIP was immunoprecipitated using a goat antibody. After fractionation by SDS–polyacrylamide gel electro- phoresis samples were western blotted for Rb or p130 as shown, (b) as for (a) except cells were infected with dl520 or dl1108 and blotted for Rb. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C- terminal-binding protein interacting protein; Rb, retinoblastoma.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Infection, Immunoprecipitation, Fractionation, Western Blot, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 10 Ad5E1A inhibits phosphorylation of CtIP. A549 cells and A549 cells stably expressing Ad512SE1A or Ad513SE1A were subjected to IR (20 Gy). Cells were harvested at the times shown and western blotted for CtIP and b-actin. Phosphorylated CtIP can be seen as a slower migrating band. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Western Blot, Binding Assay