Supporting Information

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Table 1. The cell lines tested for the TNF-dependent secretion of KRS. The cells were treated with TNF-a and TGF-b, and KRS secretion was determined as described in Materials and Methods.

Fig. 6. Dose-dependent cell binding of lysyl-tRNA synthetase (KRS) at low temperature. The indicated concentrations of KRS were treated to RAW264.7 and THP-1 cells at 4°C for 2 h, and the degree of the cell binding was determined as described in Fig. 2D.

Fig. 7. The dose-dependent effect of KRS on the proliferation of THP-1 cells. The values are the average of four independent experiments. Tryptophanyl-tRNA synthetase (WRS) and phorbol 12-myristate 13-acetate (PMA) were used as negative and positive controls, respectively.

Fig. 8. The effect of KRS on the migration of peripheral blood mononuclear cells (PBMCs). (A) PBMCs isolated from four different healthy donors were subjected to the cell migration assay by using Transwell chamber as described in Materials and Methods. KRS was added to the lower chamber at the indicated concentrations. WRS and PMA were used as negative controls. (B Left) PBMCs were loaded to the upper chamber, and 10 nM KRS or WRS was added to the lower chamber. (Right) The cells were pretreated with 10 nM KRS or WRS for 30 min and loaded to the upper chamber of Transwell. The cells were then incubated to allow the migration through the membrane, and the migrant cells were determined as described in Materials and Methods. (C) To determine the kinases involved in KRS signaling, PBMCs were pretreated with different kinase inhibitors and subjected to cell migration assay as above. Ten nanomolar KRS was added to the lower chamber. (D) The effect of cholera and pertussis toxins on the cell migration of PBMCs was determined as described in Materials and Methods.

Fig. 9. The immunohistochemical staining of KRS and RRS (arginyl-tRNA synthetase) in breast tumor and normal tissues. T and N stand for tumor and normal region in breast, respectively. We fixed human breast cancer and normal tissues in 10% formalin for 24 h, then dehydrated and embedded the issues in paraffin. We then sliced the embedded tissues (4 mm) with a microtome (Leica, Deerfield, IL) and mounted them on silane-coated slides. The mounted tissues were dewaxed, rehydrated, and equilibrated with PBS. Endogenous peroxidase was inactivated with 3% H₂O₂ for 15 min, and antigen was retrieved by microwave cooking for 5 min three times. The slides were blocked with 4% BSA for 30 min and washed with PBS containing 0.1% Tween 20 (PBST). Primary antibodies against KRS and RRS were treated to the slides for 90 min and washed with PBST three times. HRP-conjugated anti-rabbit antibody was then incubated with the slides for 30 min. We washed the slides with PBST three times, developed with DAB (3,3'-diaminobenzidine) for 7 min, and terminated the DAB development by soaking the slides into distilled water (DW) for 10 min and counterstained the nuclei with Mayer’s hematoxylin (Sigma) for 7 min. We mounted the slides after washing the slides with DW for 10 min. Photos were captured under the microscope (Nikon Eclipse 80).