Fig. 4
YTHDF2 sufficient to mediate the degradation of NR4A1 mRNA via m6A-dependent pathway. A Left: Overlap of YTHDF target genes identified by published RIP–seq and PAR-CLIP-seq in HeLa cells. Right: Venn diagram showed the candidate targets (PAR-CLIP + RIP targets) among all YTHDF paralogs. B The levels of YTHDF paralogs in TCGA-CC cohorts. Midlines indicate the median, upper and lines indicate the first and third quartiles. C The NR4A1 mRNA expression upon silencing YTHDF paralogs were analyzed by GSE134380 dataset. D-E, NR4A1 mRNA (D) and protein (E) levels were measured by RT-qPCR and western blot assay in HeLa cells. F Protein levels of NR4A1 in CC cells with overexpression wild-type (WT) or m6A recognition defective YTHDF2 (W432A and W486A) plasmids. G RIP-qPCR assay of the NR4A1 mRNA enrichment by YTHDF2 in overexpression YTHDF2 and control HeLa cells. H The stability of NR4A1 mRNA and non-methylated mRNA (PPP1R3C) upon the silencing of YTHDF2 were determined by RT-qPCR after treatment with act-D during indicated time points. I Left: The protein stability of NR4A1 was examined by western blot assay after treatment with CHX during indicated time points. Right: Quantification of protein stability. J The interaction between YTHDF2 protein and NR4A1 protein was measured via Co-IP assay. K The relative EGFP levels of co-transfected with the indicated plasmids, and normalized to the level of β-actin (left) and RFP (right) mRNA. All experiments were performed at least 3 independent times, and data are presented as means ± SD expect where otherwise specified. *P < 0.05, **P < 0.01, ***P < 0.001