Fig. 5

Knockdown of IGF2BP3 repressed angiogenesis via reading m6A modification of VEGF. a. The enrichment of IGF2BP3 in the mRNA of VEGF (VEGFA) derived from GSE92220 (crosslinking and immunoprecipitation of IGF2BP3). b. IGF2BP3 bound to VEGF transcript performed by RIP-qPCR assay. c. Obvious m6A modification of VEGF confirmed by MeRIP-qPCR, and knockdown of m6A reader METTL3 repressed its m6A modification. d. Knockdown of IGF2BP3 repressed mRNA expression of VEGF confirmed by RT-qPCR. e. The mRNA stability and degradation halftime of VEGF in HCT116 and RKO treated by Actinomycin D. f. Knockdown of IGF2BP3 repressed concentration of secreted VEGF in cell medium confirmed by ELISA analysis. g. Construction of m6A sites mutated VEGF vectors (VEGF-mut, + 2238 from the starting codon, A to C). VEGF-wt: VEGF-wild type. h. Mutation of m6A sites in VEGF abolished the binding of IGF2BP3. i. Mutation of m6A sites in VEGF (constructed in firefly reporter) repressed the luciferase expression of reporter. NC: negative control vector. j. Tube formation assay of HUVECs treated with HCT-scr, HCT-sh1, HCT-sh2, HCT-sh2 transfected pcDNA3.1-VEGF (HCT-sh2 + VEGF) derived conditional medium (CM). Quantification of tube formation assay via ImageJ (Version 1.8.0, National Institutes of Health). k. Cell invasion ability of HUVECs treated with HCT116 derived CM performed by Transwell assay. l. Cell proliferation of HUVECs treated with HCT116 derived CM performed by CCK8 assay. (**P<0.01, ***P<0.001)