Fig. 3
From: CircRNA AFF4 induced by KDM1A promotes osteogenic differentiation through FNDC5/Irisin pathway
Circ_AFF4 interacts with IGF2BP3 and stabilizes FNDC5 mRNA
(A) Immunoblot analysis of IGF2BP3 after pulldown assay showing it interacts with circ_AFF4-IGF2BP3. (B) RIP assay indicating the interaction of IGF2BP3 with circ_AFF4 in BM-MSCs. (C) FISH assay showing the localization of circ_AFF4 and IGF2BP3 protein in the cytoplasm in BM-MSCs. Scar bar = 25 μm. (D) Upper left, RNA-binding domains within IGF2BP3 protein and a summary of IGF2BP3 truncations were shown in schematic structures. Bottom Left, relative enrichment of circ_AFF4 indicating the association of circ_AFF4 with truncated IGF2BP3 compared to the control (IgG). Right, immunoblot analysis with anti-FLAG of BM-MSCs transfected with plasmids encoding FLAG-tagged WT or truncated IGF2BP3. (E) Top, UUCA motif located at exon 1-exon 6 junction site of circ_AFF4 and the RNA probe for RNA-EMSA assay were shown with a schematic illustration. Bottom, RNA-EMSA assay showed the binding ability of purified IGF2BP3 with biotin-labeled oligonucleotides containing UUCA motif from circ_AFF4. (F) Left, sequence BLAST analysis indicating that circ_AFF4 directly targets the 3′UTR of FNDC5. Right, relative enrichment of FNDC5 and circ_AFF4 showing the association of FNDC5 and circ_AFF4 junction compared to control. (G) Circ_AFF4 knockdown significantly reduced FNDC5 enrichment in BM-MSCs. (H) Luciferase activity of luciferase reporter gene with FNDC5-WT or FNDC5-mut in circ_AFF4-overexpressed or circ_AFF4-knockdown BM-MSCs. (I) The co-localization of IGF2BP3 and FNDC5 was shown FISH image. Scar bar = 25 μm. (J) Relative enrichment of FNDC5 indicating the association of FNDC5 with truncated IGF2BP3 protein. (K) RIP assay showing the association of IGF2BP3 with FNDC5 upon circ_AFF4 knockdown or overexpression. * p < 0.05, ** p < 0.001 and *** p < 0.001. Each experiment was performed at least three times independently.