Figure 1

IGF2BP2 is downregulated in cerebral AVMs and regulates angiogenesis in endothelial cells. (A) Enrichment analysis of downregulated genes between normal tissues and cerebral arteriovenous malformations. (B) Rank plot showing genes enriched in the metabolism of RNA pathway. (C) Box plot showing the expression of IGF2BP2 between cerebral AVMs and normal tissues. (D) qRT-PCR and (E) Western blot detecting the IGF2BP2 knockdown efficiency in endothelial cells. (F) Representative images and (G) statistical analysis of tube formation assay in control and IGF2BP2-deficient endothelial cells. (H) qRT-PCR and (I) Western blotting detecting the IGF2BP2 overexpression in endothelial cells. (J) Representative images and (K) statistical analysis of tube formation assay in control and IGF2BP2-overexpressing endothelial cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 2

IGF2BP2 regulates vascular development-related pathways in endothelial cells. (A) Rank plot depicting differentially expressed genes between IGF2BP2 knockdown and control endothelial cells. (B) Venn diagram showing the intersection between downregulated genes in IGF2BP2 knockdown endothelial cells and genes downregulated at the level of m6A modification in METTL3-deficient or WTAP-deficient endothelial cells. (C) Network diagram displaying the results of enrichment analysis of target genes. (D) Sankey diagram showing the genes enriched in the vascular development-related pathways. (E) Bubble diagram displaying the expression levels between control and METTL3-deficient or WTAP-deficient endothelial cells. (F) Correlation analysis of IGF2BP2 expression with LGALS8 expression in cerebral AVMs. (G) Correlation analysis of IGF2BP2 expression with RNF213 expression in cerebral AVMs.
Figure 3

IGF2BP2 regulates LGALS8 expression in endothelial cells. (A) Integrative Genomics Viewer (IGV) tracks displaying RNA-seq reads distribution in LGALS8 mRNA. (B) IGV tracks displaying MeRIP-seq reads distribution in LGALS8 mRNA. (C) qRT-PCR detecting differences in LGALS8 expression between control and IGF2BP2 knockdown endothelial cells. (D) Western blot detecting differences in LGALS8 expression between control and IGF2BP2 knockdown endothelial cells. (E) qRT-PCR detecting differences in LGALS8 expression between control and IGF2BP2-overexpressing endothelial cells. (F) Western blot detecting differences in LGALS8 expression between control and IGF2BP2-overexpressing endothelial cells. (G) The mRNA half-life of LGALS8 transcript in control and IGF2BP2-overexpressing endothelial cells. (H) qRT-PCR detecting differences in LGALS8 expression between control and IGF2BP2 knockdown endothelial cells at specific time points. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 4

LGALS8 deficiency inhibits tube formation of endothelial cells. (A) qRT-PCR and (B) Western blot detecting the efficiency of LGALS8 knockdown in endothelial cells. (C) Representative images and (D) statistical analysis of tube formation assay in control and LGALS8-deficient endothelial cells. (E) qRT-PCR and (F) Western blotting detecting LGALS8 overexpression in endothelial cells. (G) Representative images and (H) statistical analysis of tube formation assay in control and LGALS8-overexpressing endothelial cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 5

IGF2BP2 and LGALS8 regulate vascular development in the zebrafish model. (A,B) Schematic of CRISPR/Cas9-mediated generation of igf2bp2a/b crispant zebrafish and identification via RNA-sequencing. (C) Representative images of cerebrovascular structures and (D) statistical analysis of mid- and hind-brain vascular areas in control and igf2bp2a/b crispant zebrafish. (E) Representative images of cerebrovascular structures and (F) statistical analysis of mid- and hind-brain vascular areas in igf2bp2a/b crispant and rescue zebrafish. (G,H) Schematic of CRISPR/Cas9-mediated generation of lgals8a/b crispant zebrafish and identification via RNA-sequencing. (I) Representative images of cerebrovascular structures and (J) statistical analysis of mid- and hind-brain vascular areas in control and lgals8a/b crispant zebrafish. (K) Representative images of cerebrovascular structures and (L) statistical analysis of mid- and hind-brain vascular areas in lgals8a/b crispant and rescue zebrafish. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Acknowledgments
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