Fig. 4.

YTHDC2 interacts with ATG5 mRNA to promote its translation. A Expression of m⁶A readers in Huh7 detected by Western blot. B Expression of ATG5 protein detected by Western blot. C Translation efficiency of ATG5 in Huh7, calculated as: translation efficiency = ATG5 protein level/ATG5 mRNA expression level. D ActD was used to inhibit the transcription and RT-PCR was used to detected the mRNA of ATG5. E Puromycin labeling assay showed the protein synthesis in Huh7. F-G Huh7 cells were transfected with pmiR-GLO plasmids containing ATG5 CDS region for 24 h, and the translation efficiency of ATG5 was illustrated as the relative ratios between F-luc and R-luc. H RIP experiments to analyze the binding of ATG5 and YTHDC2. I The levels of m⁶A modification in ATG5 mRNA 3′-UTR, CDS and 5′-UTR were determined by MeRIP qPCR. J Diagram of the m⁶A site on ATG5 mRNA. K Schematic representation of the mutation in CDS on ATG5 mRNA. L RIP experiments to analyze the binding of ATG5 and YTHDC2. M Puromycin labeling assay showed the protein synthesis in Huh7. The mean ± SD was used as the value of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.