Fig 1: Circ_AFF4 and KDM1A were significantly increased in BM-MSCs upon osteoinduction (A) Protein expression of cell surface markers, CD14, CD45, HLA-DR, CD73, CD90 and CD105, in BM-MSCs was assessed by FACS. (B) Representative images of ARS and ALP staining of BM-MSCs cultured in osteogenic medium (OM) or normal medium (NM) for 14-days. Scar bar = 100 μm. Relative gene (C) and protein (D) expression of osteogenic markers, ALP, RUNX2, OPN and Colla 1, in BM-MSCs cultured in OM or NM for 14-days, were detected by RT-qPCR and western blot, respectively. (E) Relative gene expression of circ_AFF4 and KDM1A were assessed by RT-qPCR in BM-MSCs cultured in OM or NM after 0, 3, 7 and 14 days. (F) Relative protein expression of KDM1A was detected by western blot in BM-MSCs cultured in OM or NM after 14 days. *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: 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
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