Fig 3

Fig 3

(A) EMSA to measure the binding of SerRS^WT, SerRS^AA, or SerRS^DD with ³²P-labeled DNA fragments (27 bp) corresponding to the SerRS binding site on human VEGFA promoter. (B) Chromatin-immunoprecipitated DNA by SerRS antibody was quantified by qPCR (ChIP-qPCR) to measure the binding of SerRS^WT, SerRS^AA, or SerRS^DD on VEGFA promoter in HEK293 cells. The data were calculated as percentage of input DNA and then normalized against the SerRS^WT group (means ± SEM, n = 2, biological replicates, ****p < 0.0001, Student t test). (C) Structure model of SerRS dimer binding to VEGFA promoter DNA showing that the phosphorylation sites S101 and S241 are located near the predicted SerRS-DNA binding sites. (D, E) ChIP-qPCR to follow the binding of endogenous SerRS, c-Myc, and HIF-1α on VEGFA promoter during hypoxia in HUVEC cells (D) and HEK293 cells (E) (means ± SEM, n = 3, biological replicates, **p < 0.01, Student t test). (F) ChIP-qPCR to measure the binding of SerRS, c-Myc, and HIF-1α on VEGFA promoter in HEK293 cells stably transfected with SerRS^WT, SerRS^AA, or SerRS^DD (tag-free) under normoxia and hypoxia (12 h) conditions (means ± SEM, n = 3, biological replicates, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student t test). The expression level of the exogenous SerRS proteins (WT, AA, and DD) is similar (as shown in Fig 4A and S5 Fig). (G) qRT-PCR to measure VEGFA expression in HEK293 cells transfected with SerRS^WT or SerRS^AA under normoxia and hypoxia conditions (means ± SEM, n = 4, biological replicates, *p < 0.05, **p < 0.01, n.s., Student t test). (H) qRT-PCR to measure the effects of specific ATM inhibitor KU-55933 and specific ATR inhibitor VE-821 on hypoxia-induced VEGFA expression in HEK293 cells (means ± SEM, n = 2, biological replicates, *p < 0.05, ****p < 0.0001, Student t test). (I) ChIP-qPCR to measure the binding of SerRS to VEGFA promoter in HEK293 cells stably transfected with SerRS^WT, SerRS^AA, or SerRS^DD under normoxia or hypoxia, in the presence or absence of VE-821 (means ± SEM, n = 3, biological replicates, **p < 0.01, ***p < 0.001, Student t test). See S1 Data for quantitative data and statistical analysis. See S2 Data for original, uncropped images supporting blots and gel results. ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutated and RAD3-related; ChIP-qPCR, chromatin immunoprecipitation–quantitative real-time PCR; EMSA, electrophoresis mobility shift assay; HIF-1; hypoxia-inducible factor 1; HUVEC, human umbilical vein endothelial cell; n.s., not significant; qRT-PCR, real-time quantitative reverse transcription PCR; SerRS, seryl-tRNA synthetase; SerRS^AA, S101A/S241A; SerRS^DD, S101D/S241D; SerRS^WT, wild-type SerRS; VEGFA, vascular endothelial growth factor A.