Fig 1: KDM1A directly binds to circ_AFF4 and FNDC5 to promote FNDC5/Irisin expression (A) Schematic illustration showing binding sites between circ_AFF4 promoter and KDM1A predicted by AnimalTFDB, (B) ChIP assay showing the enrichment of BS1-5 sites of circ_AFF4 promoter in complexes precipitated with antibody against KDM1A. (C) ChIP assay showing the impact of KDM1A knockdown on KDM1A/circ_AFF4 promoter interaction. (D) Schematic illustration showing binding sites between FNDC5 promoter and KDM1A predicted by AnimalTFDB, (E) ChIP assay showing the enrichment of BS1-8 sites of FNDC5 promoter in complexes precipitated with antibody against KDM1A. (F) ChIP assay showing the impact of KDM1A knockdown on KDM1A/FNDC5 promoter interaction. Circ_AFF4, FNDC5 and KDM1A were detected by (G) RT-qPCR and (H) western blot. (I) ELISA detected Irisin expression. *p < 0.05, **p < 0.001 and ***p < 0.001. Each experiment was performed at least three times independently
Fig 2: Knockdown of KDM1A suppresses osteogenic differentiation of BM-MSCs (A) BM-MSCs were transfected with shKDM1A_1–4# or shNC and the expression level of KDM1A in shKDM1A_1–4# or shNC transfected BM-MSCs was detected using western blot. BM-MSCs that were transfected with shKDM1A or shNC were cultured in OM or NM for 14 days. Afterward, different assays were performed to assess the osteogenic differentiation. (B) Representative images of ARS and ALP staining. Scar bar = 100 µm. (C) Representative RUNX2 immunofluorescence staining image. Scar bar = 100 µm. Gene and protein expression of osteogenic markerslivergent, ALP, OPN, RUNX2 and Colla1, and circ_AFF4, FNDC5 were detected by (D) RT-qPCR and (E) western blot. (F) ELISA detected Irisin expression. *p < 0.05, **p < 0.001 and ***p < 0.001. Each experiment was performed at least three times independently.
Fig 3: Promotion of circ_AFF4 on osteogenic differentiation via FNDC5/Irisin was counteracted by IGF2BP3 downregulation (A) BM-MSCs were transfected with shIGF2BP3_1–4# or shNC and the protein expression level of IGF2BP3 in shIGF2BP3_1–4# or shNC transfected BM-MSCs was detected using western blot. BM-MSCs that transfected with lentivirus-circ_AFF4 (lv-circ_AFF4) and shNC or shIGF2BP3 and lv-circ_AFF4 or shNC and lentivirus-NC (lv-NC) were cultured in OM or NM for 14 days. Gene and protein expression of IGF2BP3 and FDNC5/Irisin were assessed by western blot (B), RT-qPCR (C) and ELISA(D). Afterward, different assays were performed to assess the osteogenic differentiation. (E) Representative images of ARS and ALP staining. Scar bar = 100 µm. (F) Representative RUNX2 immunofluorescence staining image. Scar bar = 100 µm. Gene and protein expression of osteogenic markers, ALP, OPN, RUNX2 and Colla1, were detected by (G) RT-qPCR and (H) western blot. *p < 0.05, **p < 0.001 and ***p < 0.001. Each experiment was performed at least three times independently.
Fig 4: 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.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Human Irisin/FNDC5 ELISA Kit (Colorimetric)