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. 2023 Sep 27;11:1236356. doi: 10.3389/fcell.2023.1236356

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Copyright © 2023 Singh, Walter, Elcheva, Imamura Kawasawa and Spiegelman.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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IGF2BP1 contribution to Wnt/β-catenin regulation of transcriptome in non-transformed cells. (A) MEF has been isolated from the 13.5 days old UBC-CreERT2 Igf2bp1 flox/flox mouse embryo, and Igf2bp1 knockout MEF cells were generated by treating cells with tamoxifen. (B) The Igf2bp1 knockout in MEFs was confirmed using an immunoblot assay. (C) The TOP/FOP Flash assay shows the extent of Wnt/β-Catenin signaling induction in MEF-wild type and MEF-Igf2bp1-knockout cells after Wnt3a ligand treatment. The data are means ± SD (n = 3). (D) Pie chart analysis of transcriptome expression showing significantly altered genes after Wnt/β-Catenin signaling induction in wild type MEF. (E) Bar plot showing transcriptomic expression profile of Upregulated genes shown in Figure 1D, after Wnt/β-Catenin signaling induction in Igf2bp1-knockout MEF cells. (F) Bar plot showing transcriptomic expression profile of downregulated genes shown in Figure 1D, after Wnt/β-Catenin signaling induction in Igf2bp1-knockout MEF. (G) Pie chart showing total Wnt/β-Catenin dependent transcriptomic changes dependent and independent of Igf2bp1. (H) Immunoblotting was used to confirm the enrichment of Igf2bp1 in a bead-bound fraction of Crosslinked RNA-Immunoprecipitation using Igf2bp1 antibody and control IgG isotype in iCLIP assaay. (I,J) Pie chart indicates the distribution of significantly enriched IGF2BP1 peak locations across the transcriptome in untreated (I), and Wnt3a treated MEF (J). (K) Bar graph showing the total number of significant binding peaks found on noncoding and messenger RNA in untreated and Wnt3a-treated MEF. (L) Three most enriched predicted Igf2bp1 motifs found in untreated and Wnt3a treated iCLIP assay analysis. (M) The bar diagram shows genes common between list of differentially expressed genes (DEG) found in RNA sequencing that are regulated by Wnt signaling + IGF2BP1 (Figure 3E), and list of genes found in iCLIP analysis of MEF-Wnt3a treated cells.