Figure 3.

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.