Journal: Journal for Immunotherapy of Cancer

Article Title: Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1

doi: 10.1136/jitc-2019-000277

Figure Lengend Snippet: UNE-C1-mediated activation of APCs via TLR2/6. (A) BMDCs were incubated with different CARS1 fragments for 24 hours. Costimulatory molecules were analyzed from the gated CD11c + population. CD86 expression was evaluated by flow cytometry, and IL-6 and IL-12p70 secretion in supernatants was quantified by ELISA. (B) hTLR2 and hTLR4 HEK-Blue cells, expressing SEAP reporter gene in response to NF-Kβ activity, were treated with CARS1 or UNE-C1 in a dose-dependent manner. HEK-Blue TLR2 and TLR4 activation was evaluated by measuring SEAP secretion in culture media. (C) PMA-differentiated THP-1 cells were preincubated with the indicated amount of anti-human TLR2 or anti-human TLR4 for 1 hour and treated with CARS1 or UNE-C1 for an additional 4 hours. TNF-α from supernatants of PMA-differentiated THP-1 was measured by ELISA. (D) His-tagged CARS1 or UNE-C1 were incubated with TLR2-Flag or TLR4-flag proteins. His-ab or Mock-ab bound protein G agarose was used for immunoprecipitating his-tagged proteins. (E) Reciprocal immunoprecipitation was performed using Flag-ab or Mock-ab bound protein-G agarose. His-CARS1 or -UNE-C1 was incubated with TLR2-Flag or TLR4-flag. Interactions were determined by immunoblotting (F) BMDCs from naïve and TLR2 −/− mice were treated with CARS1 or UNE-C1 for 24 hours. IL-6 and IL-12p70 levels in supernatants were quantified. (G) CARS1 and UNE-C1 were treated on hTLR2/6 and hTLR1/2. SEAP activities were measured at OD 620 nm. Data are representative of three independent experiments. Results are presented as mean±SD, and statistical significance was analyzed with Student’s t-test (***p<0.001). APC, antigen-presenting cell; BMDC, bone marrow-derived dendritic cell; CARS1, cysteinyl-tRNA synthetase 1; IL, interleukin; LPS, lipopolysaccharide; NF-Kβ, nuclear factor kappa-light-chain-enhancer of activated B cells; NS, not significant; OD, optical density; PMA, phorbol 12-myristate 13-acetate; SEAP, secreted embryonic alkaline phosphatase.

Article Snippet: TLR2 and TLR4 were purified from human embryonic kidney (HEK) 293 cells transfected with pCMV3-TLR2-flag, and pCMV3-TLR4-flag, respectively (Sino Biological).

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunoprecipitation, Western Blot, Derivative Assay

Journal: Cell reports

Article Title: Effector memory T cells induce innate inflammation by triggering DNA damage and a non-canonical STING pathway in dendritic cells

doi: 10.1016/j.celrep.2023.113180

Figure Lengend Snippet: (A) TRAF6 and STING protein expression following co-immunoprecipitation (coIP) of STING in WT BMDCs cultured with STING gt/gt T EM cells and anti-CD3 for 1 or 3 h. BMDCs stimulated with DMXAA for 1 h were used as a control for canonical STING activation. (B–E) Histograms and median fluorescence intensity (MFI) of phosphorylated IKKα/β in BMDCs (B and C) and sDCs (D and E) after 1 h of T EM with or without anti-CD3. (F) Quantified %pro-IL-1β+ WT or STING gt/gt BMDCs with or withoutTRAF6 sgRNA transfection following culture with WT T EM cells in the presence of anti-CD3 for 3 h. (G and H) IL-1β or IL-6 was measured by ELISA in the supernatants of WT or STING gt/gt BMDCs with or without TRAF6 sgRNA transfection cultured with WT T EM cells in the presence of anti-CD3. Error bars indicate SEM. In (A)–(H), 3 independent experiments; in (B)–(H), n = 5–8; two-way ANOVA.

Article Snippet: Traf6 sgRNA 5’-GAAACTCAGAGTATGTACGT-3’ , Synthego , N/A.

Techniques: Expressing, Immunoprecipitation, Cell Culture, Control, Activation Assay, Fluorescence, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Effector memory T cells induce innate inflammation by triggering DNA damage and a non-canonical STING pathway in dendritic cells

doi: 10.1016/j.celrep.2023.113180

Figure Lengend Snippet:

Article Snippet: Traf6 sgRNA 5’-GAAACTCAGAGTATGTACGT-3’ , Synthego , N/A.

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, In Vivo, Recombinant, Red Blood Cell Lysis, Protease Inhibitor, Lysis, SYBR Green Assay, Staining, Reverse Transcription, Single Cell Gel Electrophoresis, Cell Isolation, Bicinchoninic Acid Protein Assay, RNA Sequencing, Software, Real-time Polymerase Chain Reaction, Inverted Microscopy, Microscopy, Autoradiography

Journal: Innate Immunity

Article Title: Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

doi: 10.1177/1753425920927479

Figure Lengend Snippet: Diagram of the TLR4 signalling pathway.

Article Snippet: PHY-028-GFP-TLR4 (WT), PHY-028-GFP-TLR4 (K694R), pcDNA3.0-Flag-MD2, pcDNA3.0-CD14, pNF-κB-luc, pRL-SV40-N and pcDNA3.0-Flag-MyD88 were bought from Hanyin Biotechnology Limited Company (Shanghai, PR China).

Techniques:

Journal: Innate Immunity

Article Title: Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

doi: 10.1177/1753425920927479

Figure Lengend Snippet: The K694R polymorphism does not affect TLR4 expression levels. (a) Diagram of wild type (WT) and K694R (K694G) TLR4 plasmid vector. (b) mRNA expression levels for the genes TLR4 in human embryonic kidney 293T (HEK293T) cells transiently transfected with WT or K694R TLR4 plasmid or empty vector as control, treated with or without LPS for 6 h. (c) Western blot (WB) analysis of TLR4 expression levels in HEK293T cells transiently transfected with WT, K694R TLR4 plasmid or empty vector treated with or without LPS for 6h. (d) Quantification of TLR4 expression levels in (c). Graphs are the mean ± SD, n = 6, * P < 0.05, Student’s t -test.

Article Snippet: PHY-028-GFP-TLR4 (WT), PHY-028-GFP-TLR4 (K694R), pcDNA3.0-Flag-MD2, pcDNA3.0-CD14, pNF-κB-luc, pRL-SV40-N and pcDNA3.0-Flag-MyD88 were bought from Hanyin Biotechnology Limited Company (Shanghai, PR China).

Techniques: Expressing, Plasmid Preparation, Transfection, Control, Western Blot

Journal: Innate Immunity

Article Title: Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

doi: 10.1177/1753425920927479

Figure Lengend Snippet: The K694R polymorphism does not affect cell-surface TLR4 expression. (a) Representative flow cytometric identification of HEK293T cells transiently transfected with K694R or WT TLR4 plasmid, treated with or without LPS for 6 h, respectively, gated on GFP+ cells. The cell-surface TLR4 was stained with the anti-TLR4 Ab. Anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor® 647 Conjugate) was used as the second Ab. (b) Quantification of cell-surface TLR4 stained with anti-TLR4 Ab in GFP+ cells in the Q2 quadrant. Graphs are the mean ± SD, n = 6, * P < 0.05, Student’s t -test.

Article Snippet: PHY-028-GFP-TLR4 (WT), PHY-028-GFP-TLR4 (K694R), pcDNA3.0-Flag-MD2, pcDNA3.0-CD14, pNF-κB-luc, pRL-SV40-N and pcDNA3.0-Flag-MyD88 were bought from Hanyin Biotechnology Limited Company (Shanghai, PR China).

Techniques: Expressing, Transfection, Plasmid Preparation, Staining

Journal: Innate Immunity

Article Title: Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

doi: 10.1177/1753425920927479

Figure Lengend Snippet: The K694R polymorphism does not affect total TLR4 levels. (a) Representative flow cytometric identification of HEK293T cells transiently transfected with K694R or WT TLR4 plasmid, treated with or without LPS for 6 h, respectively, gated on GFP+ cells. Total TLR4 was stained with anti-TLR4 Ab. Anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor® 647 Conjugate) was used as the second Ab. (b) Quantification of total TLR4 stained with anti-TLR4 Ab in GFP+ cells in the Q2 quadrant. Graphs are the mean ± SD, n = 6, * P < 0.05, Student’s t -test.

Article Snippet: PHY-028-GFP-TLR4 (WT), PHY-028-GFP-TLR4 (K694R), pcDNA3.0-Flag-MD2, pcDNA3.0-CD14, pNF-κB-luc, pRL-SV40-N and pcDNA3.0-Flag-MyD88 were bought from Hanyin Biotechnology Limited Company (Shanghai, PR China).

Techniques: Transfection, Plasmid Preparation, Staining

Journal: Innate Immunity

Article Title: Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

doi: 10.1177/1753425920927479

Figure Lengend Snippet: The K694R polymorphism does not affect TLR4 interaction with myeloid differentiation factor 2 (MD2). (a) HEK293T cells transiently transfected with WT or K694R TLR4, CD14 and Flag-MD2 plasmids, treated with LPS, were subjected to co-immunoprecipitation with anti-TLR4 Ab, and analysed by WB with anti-Flag Ab. (b) Densitometric quantification of the data shown in (a). The results are presented as ratios of MD2 and TLR4. Graphs are the mean ± SD, n = 3, * P < 0.05, Student’s t -test.

Article Snippet: PHY-028-GFP-TLR4 (WT), PHY-028-GFP-TLR4 (K694R), pcDNA3.0-Flag-MD2, pcDNA3.0-CD14, pNF-κB-luc, pRL-SV40-N and pcDNA3.0-Flag-MyD88 were bought from Hanyin Biotechnology Limited Company (Shanghai, PR China).

Techniques: Transfection, Immunoprecipitation

Journal: Innate Immunity

Article Title: Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

doi: 10.1177/1753425920927479

Figure Lengend Snippet: The K694R polymorphism impairs the ability of TLR4 to recruit MyD88. (a) HEK293T cells transiently transfected with WT or K694R TLR4, CD14, MD2 and Flag-MyD88 plasmids, treated with LPS, were subjected to co-immunoprecipitation with anti-TLR4 Ab, and analysed by WB with anti-Flag Ab. (b) Densitometric quantification of the data shown in (a) The results are presented as ratios of MyD88 and TLR4. Graphs are the mean ± SD, n = 3, * P < 0.05, Student’s t -test.

Article Snippet: PHY-028-GFP-TLR4 (WT), PHY-028-GFP-TLR4 (K694R), pcDNA3.0-Flag-MD2, pcDNA3.0-CD14, pNF-κB-luc, pRL-SV40-N and pcDNA3.0-Flag-MyD88 were bought from Hanyin Biotechnology Limited Company (Shanghai, PR China).

Techniques: Transfection, Immunoprecipitation

Journal: Innate Immunity

Article Title: Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

doi: 10.1177/1753425920927479

Figure Lengend Snippet: The K694R polymorphism impairs NF-κB activation and production of TNF-α, IL-1β, IL-6 and IL-18. (a) HEK293T cells transiently transfected with WT or K694R TLR4 plasmids were co-transfected with Flag-MD2, CD14, pNFκB-luc and pRL-SV40-N. Cells were treated with LPS for 6 h, and cell lysates were analysed for firefly and Renilla luciferase activities. Graphs are the mean ± SD, n = 6, **** P < 0.0001, Student’s t -test. (b) mRNA expression levels for the genes TNF-α, IL-1β, IL-6 and IL-18 in HEK293T cells transiently transfected with WT or K694R TLR4 plasmids and treated with LPS for 6 h were measured by quantitative RT-PCR. Graphs are the mean ± SD, n = 6, * P < 0.0001, Student’s t -test.

Article Snippet: PHY-028-GFP-TLR4 (WT), PHY-028-GFP-TLR4 (K694R), pcDNA3.0-Flag-MD2, pcDNA3.0-CD14, pNF-κB-luc, pRL-SV40-N and pcDNA3.0-Flag-MyD88 were bought from Hanyin Biotechnology Limited Company (Shanghai, PR China).

Techniques: Activation Assay, Transfection, Luciferase, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Oxy210, a Semi-Synthetic Oxysterol, Exerts Anti-Inflammatory Effects in Macrophages via Inhibition of Toll-like Receptor (TLR) 4 and TLR2 Signaling and Modulation of Macrophage Polarization

doi: 10.3390/ijms23105478

Figure Lengend Snippet: Inhibition of TLR4 and TLR2 agonist-induced inflammatory gene expression by Oxy210 in RAW264.7 macrophages. RAW264.7 cells were pretreated with Oxy210 (5 μM) in DMEM containing 0.1% FBS for 24 h followed by the addition of TLR4 agonist, monophosphoryl lipid A (MPLAs) (1 μg/mL), or TLR2 agonist, CU-T12-9 (1 μg/mL), in the absence or presence of Oxy210 (5 μM). After 24 h, RNA was extracted and analyzed by Q-RT-PCR for the expression of the genes as indicated and normalized to Oaz1 expression. Data from a representative experiment are reported as the mean of triplicate determinations ± SD (** p < 0.01 vs. control; ## p < 0.01 vs. MPLAs; @ p < 0.05 vs. CU-T12-9).

Article Snippet: To further confirm the inhibitory effect of Oxy210 on TLR signaling, an in vitro activity assay was performed using proprietary TLR4 Leeporter TM Luciferase Reporter-HeLa cell line or TLR2 Leeporter TM Luciferase Reporter-HEK293 cell line (Abeomics, Inc., San Diego, CA, USA).

Techniques: Inhibition, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Oxy210, a Semi-Synthetic Oxysterol, Exerts Anti-Inflammatory Effects in Macrophages via Inhibition of Toll-like Receptor (TLR) 4 and TLR2 Signaling and Modulation of Macrophage Polarization

doi: 10.3390/ijms23105478

Figure Lengend Snippet: Inhibition of TLR4, TLR2, and AP-1 driven luciferase reporter activity by Oxy210. TLR4 Leeporter TM Luciferase Reporter-HeLa cells, TLR2 Leeporter TM Luciferase Reporter-HEK293 cells, and AP-1 Leeporter TM Luciferase Reporter-HEK293 cells were treated with TLR4 agonist, LPS (25 ng/mL), TLR2 agonist, Pam3CSK4 (1 μg/mL), or AP-1 activator, PMA (100 ng/mL), respectively, for 6 h in the presence or absence of Oxy210 at the concentrations indicated. Luciferase activity was measured and normalized to the Renilla luciferase activity. Data reported as % activity calculated from triplicate wells per Oxy210 dose ± SD as described in Materials and Methods.

Article Snippet: To further confirm the inhibitory effect of Oxy210 on TLR signaling, an in vitro activity assay was performed using proprietary TLR4 Leeporter TM Luciferase Reporter-HeLa cell line or TLR2 Leeporter TM Luciferase Reporter-HEK293 cell line (Abeomics, Inc., San Diego, CA, USA).

Techniques: Inhibition, Luciferase, Activity Assay