FIGURE

Fig 3

ID
ZDB-FIG-210108-3
Publication
Shi et al., 2020 - Phosphorylation of seryl-tRNA synthetase by ATM/ATR is essential for hypoxia-induced angiogenesis
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Fig 3

ATM/ATR-caused SerRS phosphorylation attenuates its binding to <italic>VEGFA</italic> promoter, which allows c-Myc and HIF-1 to activate <italic>VEGFA</italic> transcription.

(A) EMSA to measure the binding of SerRSWT, SerRSAA, or SerRSDD 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 SerRSWT, SerRSAA, or SerRSDD on VEGFA promoter in HEK293 cells. The data were calculated as percentage of input DNA and then normalized against the SerRSWT 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 SerRSWT, SerRSAA, or SerRSDD (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 SerRSWT or SerRSAA 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 SerRSWT, SerRSAA, or SerRSDD 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; SerRSAA, S101A/S241A; SerRSDD, S101D/S241D; SerRSWT, wild-type SerRS; VEGFA, vascular endothelial growth factor A.

Expression Data

Expression Detail
Antibody Labeling
Phenotype Data

Phenotype Detail
Acknowledgments
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