Figure 1.

Effect of secreted CARS1 on TNF-α secretion from macrophages (A) CARS1 secretion was tested by incubating HCT116 cells under different conditions, including SF, TNF-α (10 ng/mL), tunicamycin (1 µg/mL), arsenite (12.5 µM), CoCl₂ (100 µM), Wnt3a (200 ng/mL), IL-2 (10 ng/mL), IL-4 (10 ng/mL), VEGF (20 ng/mL), BMP4 (50 ng/mL), PDGF (100 ng/mL), EGF (100 ng/mL) FGF-2 (50 ng/mL), FGF-4 (50 ng/mL), IGF (50 ng/mL), and glutamine-free conditions for 24 hours. The amount of CARS1 secreted in the medium and WCL was detected. (B) HCT116 cells were treated with tunicamycin (Tunica) in dose-dependent and time-dependent manners. Proteins in the medium were precipitated and detected by an antibody against CARS1. (C) HCT116 cells were treated with TNF-α in dose-dependent and time-dependent manners to detect secreted CARS1. (D, E) CARS1 and BSA were conjugated with Alexa-Fluor 647 and treated for 30 min to different cell types. CARS1-bound cells were visualized and analyzed by confocal microscopy (D) and flow cytometry (E), respectively. (F) RAW264.7 cells were treated with either 100 nM of CARS1 or KARS1 for 4 hour. Boiled CARS1 and KARS1 were used for negative controls. (G) The production of TNF-α was measured by ELISA after treating 100 nM of CARS1 on PMA-differentiated THP-1 cells. CARS1 was preincubated with proteinase K (50 µg/mL) or boiled for 1 hour. Before adding CARS1, some cells were preincubated with polymyxin B (10 µg/mL) for 1 hour. 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). BSA, bovine serum albumin; BMP4, bone morphogenetic protein 4; CARS1, cysteinyl-tRNA synthetase 1; IL, interleukin; LPS, lipopolysaccharide; PDGF, platelet-derived growth factor; PMA, phorbol 12-myristate 13-acetate; SF, serum-free; TNF-α, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor; WCL, whole-cell lysate.