Fig. 4.
APOE mRNA is regulated by FTO-mediated m6A modification through the m6A reader IGF2BP2. a Differentially expressed genes between si-NC group and si-FTO group in K1 cells as determined by RNA-sequencing. b Peak profiles in m6A modification after FTO knockdown in K1 cells as shown by MeRIP-sequencing. c Distribution of m6A peaks in mRNA transcripts of K1 cells. d The m6A consensus motif present in K1 cells. e Differentially expressed genes accompanied with elevated m6A peaks after FTO knockdown were filtered and APOE was identified as the direct target of FTO. f m6A modification of APOE mRNA was visualized by Integrative Genomics Viewer (IGV) software after FTO knockdown. The significantly increased m6A peak is indicated by red rectangles. g-j APOE mRNA and protein expression level as determined by qRT-PCR and western blotting after FTO knockdown or overexpression in PTC cells. k Relative APOE mRNA expression as determined by qRT-PCR after transfection with si-NC or si-FTO and simultaneous treatment with or without 3-deazaadenosine (DAA, a global methylation inhibitor) in K1 cells. l Direct binding between m6A antibody and APOE mRNA was validated by agarose electrophoresis and MeRIP assays in K1 cells. m Relative enrichment of m6A antibody and APOE mRNA as determined by MeRIP-qPCR after FTO knockdown in K1 cells. n-o Relative luciferase activity of APOE-Wt and APOE-Mut as determined after FTO knockdown or overexpression in PTC cells. p Half-life (t1/2) of APOE mRNA as determined by qRT-PCR after FTO knockdown in K1 cells. q Relative expression of APOE mRNA as determined by qRT-PCR after IGF2BP1–3 knockdown in K1 cells. r Half-life (t1/2) of APOE mRNA as determined by qRT-PCR after IGF2BP2 knockdown in K1 cells. s Direct binding between IGF2BP2 protein and APOE mRNA as validated by agarose electrophoresis and RIP assays in K1 cells. t Relative enrichment of IGF2BP2 protein and APOE mRNA as determined by RIP-qPCR after FTO knockdown in K1 cells. *P < 0.05, **P < 0.01