Fig. 4

YTHDF1 enhances ANGPTL4 translation via an m⁶A-dependent manner. A Distribution of m⁶A-binding peaks (MeCLIP-seq from GSE147440 and MeRIP-seq) across the ANGPTL4 transcript. B Genome browser snapshot of the m⁶A residue in ANGPTL4 (GSE147440). The red box surrounding the “A” indicates the m⁶A site (chr19: 8,374,164), with the C-to-T mutations at the downstream “C” depicted in the reads (blue bars) aligning to that locus. C Schematic diagram of the procedure for screening ANGPTL4 mRNA m⁶A readers using RNA pull-down and mass spectrometry with ANGPTL4 m⁶A site-specific methylated single‐stranded RNA (ss‐m⁶A) or unmethylated control RNA (ss‐A) probes. D Candidate proteins binding to the ANGPTL4 ss-m⁶A probes identified by mass spectrometry. E Western blot analysis of YTHDF1 in MDA-MB-468 cells following the RNA pull-down assay. F YTHDF1-specific RIP-derived RNA from MDA-MB-231 and MDA-MB-468 cells was measured by qPCR. G Relative RNA levels of ANGPTL4 in MDA-MB-231 and MDA-MB-468 cells upon YTHDF1 knockdown. H Western blot analysis detected the protein levels of ANGPTL4 in MDA-MB-231 and MDA-MB-468 cells upon YTHDF1 knockdown. I Polysome profiling of MDA-MB-468 cells following YTHDF1 knockdown (left), and relative ANGPTL4 mRNA distribution in each ribosome fraction analyzed by qPCR (right). J Schematic description of wild-type (YTHDF1-WT) and mutant (YTHDF1-Mut) constructs of YTHDF1. K Western blot analysis of ANGPTL4 in TNBC cells transfected with YTHDF1-WT or YTHDF1-Mut. L Flag-specific RIP-derived RNA in MDA-MB-468 cells was measured by qPCR. M Schematic description of wild-type (ANGPTL4-WT) and mutant (ANGPTL4-Mut) constructs of ANGPTL4. N Western blot analysis of ANGPTL4 expression in MDA-MB-231 and MDA-MB-468 cells transfected with the empty vector, Flag-tagged YTHDF1-WT/-Mut, and HA-tagged ANGPTL4-WT/-Mut. *P < 0.05, **P < 0.01, ***P < 0.001, NS, no significance