Fig. 4

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.