SerRSAA exhibits stronger activity than SerRSWT in inhibiting tumor angiogenesis and tumor growth in mice.(A–C) Tumor xenografts formed by the original (control) or engineered MDA-MB-231 cells were analyzed by monitoring the growth curves (A) and comparing the size of the xenografts at end point (22 days post implantation) (B) with quantifications (C). (D–F) The dissected MDA-MB-231 breast cancer xenografts were analyzed by western blots for SerRS, HIF-1α, VEGFA, Cdh5, and β-actin protein levels (D), and the levels were quantified for VEGFA (E) and Cdh5 (F). (G–J) Tumor angiogenesis in MDA-MB-231 xenografts was analyzed by immunofluorescent staining of VEGFA (G) and Cdh5 (I) and quantified by measuring the fluorescent signal intensities (H, J). (K, L) IP and western blot analysis to confirm SerRS phosphorylation in tumor xenografts. IP was performed with anti-SerRS antibody from rabbit before phosphorylated SerRS was detected with a specific antibody against p-S*Q (K) and quantified by measuring the band intensities (L). Data are shown as means ± SEM, n = 5 mice, *p < 0.05, **p < 0.01, ***p < 0.001, n.s., Student t test). See S1 Data for quantitative data and statistical analysis. See S2 Data for original, uncropped images supporting blots and gel results. HIF-1α; hypoxia-inducible factor 1α; IP, immunoprecipitation; n.s., not significant; SerRS, seryl-tRNA synthetase; SerRSAA, S101A/S241A; SerRSDD, S101D/S241D; SerRSWT, wild-type SerRS; VEGFA, vascular endothelial growth factor A.
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