Fig. 4

FTO-IT1 enhanced the interaction between ILF2 and ILF3 protein and stabilized FTO mRNA by strengthening the binding between ILF2/ILF3 complex and FTO mRNA. A Biotin-labeled RNA pull-down followed by Western blot showed the interaction between FTO-IT1 and ILF2/ILF3 protein in HepG2/MHCC97H cells. B RIP assay was applied using the anti-ILF2/ILF3 antibody and IgG antibody. The detection of FTO-IT1 (upper) was performed using specific primers in HepG2 cells. Then the PCR products were run on a 2% agarose gel (lower). C Western blot of ILF2/ILF3 pulled down by full-length (FL: 1–870 nt) or a series of FTO-IT1 truncates (Del 1–5) or antisense in HepG2 cells. D, E Co-IP of endogenous ILF2 and ILF3 with transfected siNC, siFTO-IT1 (D), empty vector or overexpression FTO-IT1 plasmid (E) in HepG2 cells. F The mRNA (left) and protein (right) levels of FTO in ILF2 knockdown or ILF3 knockdown HepG2/MHCC97H cells compared with control. G HepG2/MHCC97H cells transfected with siNC, siFTO-IT1, and then treated with ActD (5 μg/mL) for 0, 2, 4, 6 and 8 h, respectively, followed by qRT-PCR assays for FTO mRNA. H Schematic illustration showed the binding between ILF2/ILF3 and AREs in 3’UTR of FTO mRNA using online tool AREsite2 (http://rna.tbi.univie.ac.at/AREsite2/welcome). I Western blot of ILF2/ILF3 pulled down by 3’UTR of FTO or antisense (3’UTR-AS) in HepG2 cells transfected with siNC or siFTO-IT1. J RIP assays were applied using the anti-ILF2/ILF3 antibody and IgG antibody in HepG2 cells transfected with siNC or siFTO-IT1. qRT-PCR was used to detect the corresponding enrichment of 3’UTR of FTO mRNA. K HepG2 cells transfected with siNC, siFTO-IT1, or co-transfected with overexpression ILF2 or overexpression ILF3 plasmid and treated with ActD for corresponding hours followed by qRT-PCR assays for FTO mRNA. L The mRNA (left) and protein (right) levels of FTO in HepG2/MHCC97H cells transfected with siNC, siFTO-IT1 or co-transfected with overexpression ILF2 or overexpression ILF3 plasmid