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. 2021 Jul 3;40(33):5168–5181. doi: 10.1038/s41388-021-01916-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — A RNA pulldown was performed with biotinylated MTA1. Immunoblotting of YTH family and IGF2BP family m⁶A readers in cell lysate (Ly), biotinylated full-length MTA1 and beads only (NC) in HCT116 cells (up). Immunoblotting of IGF2BP2 with lysate (Ly), biotinylated full-length MTA1 and beads only (NC) in HCT116 and DLD1 cells by RNA pulldown assay (down). B Immunoblotting of lysate (Ly), MTA1 full length (#1), MTA1 CDS region (#2), CDS m⁶A motif mutant MTA1 (#3), MTA1 3’UTR region (#4), 3’UTR m⁶A motif mutant MTA1 (#5) and beads only (NC) by RNA pulldown assay in HCT116 and DLD1 cells. C RIP assays showed the direct binding between IGF2BP2 protein and MTA1 mRNA in HCT116 and DLD1 cells. Agarose electrophoresis (up) and qPCR analysis (down). D RIP assays demonstrating the enrichment of IGF2BP2 protein bound MTA1 mRNA in siNC versus siFTO HCT116 and DLD1 cells. E The correlation between MTA1 and IGF2BP2 RNA expression levels in TCGA dataset for COAD and READ assessed by using the GEPIA online tool (http://gepia. cancer-pku.cn/). F The correlation between MTA1 and IGF2BP2 RNA levels in 78 CRC specimens from SYSUCC. G Immunoblotting assay of MTA1, FTO and IGF2BP2 protein levels in HCT116 cells with siNC, FTO knockdown only (siFTO#1), IGF2BP2 knockdown only (siIGF2BP2#1, siIGF2BP2#2) and both FTO and IGF2BP2 knockdown (siFTO#1 + siIGF2BP2#1, siFTO#1 + siIGF2BP2#2). Data in (C, D, and G) are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).