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. 2021 Apr 15;11(4):1428–1445.

Figure 4.

Figure 4

Binding of RNA m6A by IGF2BP3 enhanced stability of ABCB1 mRNA. A. Primers for m6A RIP-qPCR displayed in the structure of EEF1A mRNA and ABCB1 mRNA (upper). m6A RIP-qPCR analysis of ABCB1 mRNA in HCT8/T and HCT8 cells (lower). m6A RIP was performed using mRNAs samples extracted from HCT8/T and HCT8 cells with anti-m6A antibody, and expression level of m6A modified mRNAs was determined by qRT-PCR. Input was used for normalization. B. Western blotting (upper) and RT-qPCR (lower) analysis of RIP assays in HCT8/T and HCT8 cells. IGF2BP3-associated mRNAs were isolated from HCT8/T and HCT8 cells by IP using anti-IGF2BP3 antibody. IgG was used for normalization. Fold enrichment of ESR2, IGF2 and m6A-modified ABCB1 mRNA bound to IGF2BP3 was measured by qRT-PCR. Primers for the m6A modification region of ABCB1 mRNA is the same as that in m6A RIP-qPCR assay. C. Western blotting (upper) and RT-qPCR (lower) analysis of RIP assays in HCT8/T cells treated with siMETTL3 or siMETTL14 for 48 h. IGF2BP3-associated ABCB1 mRNAs were isolated from HCT8/T cells with or without siRNA by IP using anti-IGF2BP3 antibody. IgG was used for normalization. Fold enrichment of m6A-modified ABCB1 mRNA bound to IGF2BP3 was determined by qRT-PCR. Primers of the m6A modification region of ABCB1 mRNA were the same as those used in m6A RIP-qPCR. D, E. Western blotting and qRT-PCR analysis of ABCB1 expression in HCT8/T cells treated with siMETTL3 or siMETTL14 for 48 h. F. Western blotting analysis performed in HCT8 cells with IGF2BP3 overexpression and control HCT8 cells or IGF2BP3 knockdown and control HCT8/T cells upon METTL3 silencing. G. Remaining ABCB1 mRNA in IGF2BP3-knockdown or control HCT8/T cells treated with 5 μg/ml Act-D at 0, 3, or 6 h (left), and remaining ABCB1 mRNA in HCT8/T cells treated with siMETTL3 or siMETTL14 for 48 h, and then with 5 μg/ml Act-D at 0, 3, or 6 h (right). Densitometry analysis of protein bands was performed using Image J. Data are shown as mean ± SD of n = 3 independent assays. n.s, P >0.05, *, P <0.05; **, P < 0.01; ***, P < 0.001 (Student’s t-test).