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: 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