Fig. 5.

Stabilization of TCF7L2 mRNA by hnRNPA2B1 and MIR100HG is m⁶A dependent. a Top, immunoblots of METTL3 showing the silencing efficiency of siRNAs against METTL3. Bottom, qPCR analysis of TCF7L2 expression in CC-CR and HCT116 cells after METTL3 knockdown. n = 3 independent biological replicates. b Analysis of MeRIP assays detecting TCF7L2 mRNA retrieved by a m⁶A antibody in METTL3-silenced CC-CR and HCT116 cells. n = 3 independent biological replicates. c Top, graphic illustration of the five high-confidence m⁶A sites predicted by SRAMP in TCF7L2 mRNA. Nucleotide positions are numbered with respect to the transcriptional start site of TCF7L2. Bottom, analysis of MeRIP assays detecting TCF7L2 mRNA retrieved by a m⁶A antibody around the five high-confidence m⁶A sites in CC-CR and HCT116 cells. n = 3 independent biological replicates. d Assessment of RIP assays detecting TCF7L2 mRNA retrieved by a hnRNPA2B1 antibody or by IgG in CC-CR and HCT116 cells. n = 2 independent biological replicates. e, f Assessment of RIP assays detecting TCF7L2 mRNA retrieved by a hnRNPA2B1 antibody or by IgG in CC-CR and HCT116 cells after METTL3 knockdown (e) or MIR100HG exon 4 knockout (f). n = 2 independent biological replicates. g Top, schematic representation of the S1m-mediated in vivo pull-down assay. Bottom, immunoblot for hnRNPA2B1 after in vivo expressed WT or mutant S1m tagged TCF7L2 mRNA was pulled down by streptavidin beads. Representative of three independent experiments. h, i Luciferase activity of WT (TCF7L2-WT) or mutated (TCF7L2-Mut) TCF7L2 reporters in HCT8 (h) and HEK293T (i) cells with ectopically expressed hnRNPA2B1. n = 2 independent biological replicates. j, k Luciferase activity of TCF7L2-WT or TCF7L2-Mut reporters in HCT8 (j) and HEK293T (k) cells with ectopically expressed hnRNPA2B1, MIR100HG ASO or METTL3 siRNA. n = 2 independent biological replicates. ***P < 0.001, **P < 0.01, *P < 0.05. Data represent mean ± s.d., n.s., not significant