Figure 4: Effects of WARS2 on endothelial cell viability and oxygen consumption.

(a) SiRNA-mediated inhibition of WARS2 in endothelial cells (ECs) results in multiple cells with two or more incompletely separated nuclei. Left panel, nuclei (arrow heads); right panel, nuclei (blue) and actin (green). Scale bar=50 μm. (b) FACS analysis of the cell cycle in proliferating ECs treated with non-targeting siRNA (siNT) or siRNA against WARS2 (siWARS2) shows a reduction in ECs in the S phase and an increased number in the G2/M phase with siW2. The experiment was repeated (n=3) with similar results (c) FACS analysis of caspase-3/7 activation and cell death over a 72-h time course. ECs were transfected with non-targeting siRNA (siNT) or siRNA against WARS2 (siWARS2). The experiment was repeated with similar results (and see Supplementary Fig. 14). (d) Immunoblot (IB) of COX11 and Flag in Cox11 immunoprecipitates (IP: COX11) and of whole cell lysates (WCL) from cells expressing either vector alone (V), Flag-tagged wild-type WARS2 (WT) or Flag-tagged mutant WARS2(L53F) (L53F). (e) Oxygen consumption rates (OCRs) in ECs transfected with non-targeting siRNA (siNT, blue boxes) or siRNA against WARS2 (siWARS2, red circles). A representative experiment is shown; the experiment repeated 5 times with similar results. (f) Quantification of basal (upper panel) and maximal (lower panel) OCRs. n=3, t-test. **, P<0.01. ***, P<0.001.