Figure 3
From: Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis
High concentrations of BC194 are required to stimulate the unfolded protein response and apoptosis in endothelial cells.
(a) Effects of BC194 on proliferation. HUVECs were exposed to the indicated concentrations of BC194 and relative proliferation was quantified over time using an alamarBlue® assay (a measure of mitochondrial oxidase activity); mean ± s.e.m., n = 3, *P < 0.05 (one-way ANOVA, Tukey test). (b) Effects of BC194 on nascent protein synthesis. HUVECs were exposed to the indicated concentrations of BC194 and new protein synthesis was detected using a Click-iT® metabolic labeling kit. Proteins were separated by SDS-PAGE and visualized using streptavidin-HRP. Cycloheximide (50 μM) was used as a control for complete inhibition of protein synthesis. (c–f) HUVECs grown in full serum media were exposed to the indicated concentrations of BC194, followed by Western Blot of cell extracts using antibodies recognizing phospho-eIF2α (c), cleaved caspase-3 (d) and β-actin or β-tubulin as a loading control. (e,f) Quantification of phospho-eIF2α and c-caspase-3 relative to the loading controls; mean ± s.e.m., n = 3, *P < 0.05 (one-way ANOVA, Tukey test).
