Fig. 4

IGF2BP3 regulated cell cycle and proliferation via reading m6A modification of Cyclin D1. a. The enrichment of IGF2BP3 in the mRNA of Cyclin D1 (CCND1) derived from GSE92220 (crosslinking and immunoprecipitation of IGF2BP3). b. The enrichment of IGF2BP3 in the mRNA of CCND1, c-Myc, CDK2 and CDK6 performed by RIP-qPCR assay. c-Myc was a known target of IGF2BP3 and worked as positive control. c. The m6A modification site of CCND1 predicted by SRAMP website tools based on sequence-derived features, and primers designed for MeRIP-qPCR assay. d. Obvious m6A modification of CCND1 confirmed by MeRIP-qPCR, and knockdown of m6A reader METTL3 repressed its m6A modification. e. Knockdown of IGF2BP3 repressed mRNA expression of CCND1 confirmed by RT-qPCR. f. The mRNA stability and degradation halftime of CCND1 in HCT116 and RKO treated by Actinomycin D. g. Knockdown of IGF2BP3 repressed protein expression of Cyclin D1 confirmed by Western Blotting analysis. h. Overexpression of Cyclin D1 (transfection of pCDNA3.1-Cylin D1) in HCT-sh1 rescued cell cycle arrest. (***Compare between percentage of G0/G1 phase, P<0.001; ^###Compare between percentage of S phase, P<0.001) (i). Overexpression of Cyclin D1 (transfection of pCDNA3.1-CCND1) in HCT-sh1 rescued inhibited DNA replication. (**P<0.01, ***P<0.001)