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Fig. 5 | Molecular Cancer

Fig. 5

From: Interaction of lncRNA MIR100HG with hnRNPA2B1 facilitates m6A-dependent stabilization of TCF7L2 mRNA and colorectal cancer progression

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

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