Figure 1. TARS is secreted by endothelial cells in response to VEGF and TNF-α.

(a) HUVECs were treated with VEGF or TNF-α (50 ng/ml) where indicated. After 6 h the level of TARS in the supernatant was determined by ELISA. Graph represents mean ± s.e.m.; n = 3, *P < 0.05 (Student's t-test). (b) Cell membrane integrity for the experiments in (a and c) was confirmed using the lactate dehydrogenase assay CytoTox-ONE^TM. Numbers represent mean percent cytotoxicity ± s.e.m. relative to a lysis control, n = 3, *P < 0.05 (Student's t-test). (c) HUVECs grown on a 10 cm dish were exposed to 50 ng/ml of VEGF or TNF-α in 0% serum EGM-2 media for 16 h. Shown is a representative TARS Western blot of cell lysates and media samples, n = 4. Media was concentrated 20-fold to accommodate 25% onto the gel and compared to 5% of the cell lysate. Purified TARS (50 fmol) was used to gauge TARS concentration within samples (See Supplementary Fig. S1). β-tubulin was measured as a loading and lysis control. (d) VEGF and TNF-α do not induce TARS transcription. HUVECs were exposed to 50 ng/ml of VEGF or TNF-α followed by RNA extraction and RT-qPCR to measure TARS mRNA levels. Shown are mean Rq values ± s.e.m. relative to a β-2 microglobulin control; n = 3, n.s. (Student's t-test). (e) TARS does not induce VEGF secretion. HUVECs were exposed to the indicated concentrations of purified recombinant human TARS for 24 h and the level of VEGF in the supernatant determined by ELISA; mean ± s.e.m., n = 3, n.s. (Student's t-test).