Journal: Molecular Medicine

Article Title: CircRNA AFF4 induced by KDM1A promotes osteogenic differentiation through FNDC5/Irisin pathway

doi: 10.1186/s10020-022-00557-7

Figure Lengend Snippet: 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.

Article Snippet: The Irisin levels in BM-MSCs were measured using ELISA kits (NBP3-08117, Novus Biologicals, Littleton, CO, USA) based on the manufacturer’s protocol.

Techniques: Transfection, Expressing, Western Blot, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence

Journal: Molecular Medicine

Article Title: CircRNA AFF4 induced by KDM1A promotes osteogenic differentiation through FNDC5/Irisin pathway

doi: 10.1186/s10020-022-00557-7

Figure Lengend Snippet: 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.

Article Snippet: The Irisin levels in BM-MSCs were measured using ELISA kits (NBP3-08117, Novus Biologicals, Littleton, CO, USA) based on the manufacturer’s protocol.

Techniques: Knockdown, Transfection, Expressing, Western Blot, Cell Culture, Staining, Immunofluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Bioengineered

Article Title: MicroRNA-665-3p exacerbates nonalcoholic fatty liver disease in mice

doi: 10.1080/21655979.2021.2017698

Figure Lengend Snippet: miR-665-3p antagomir activates AMPKα via increasing FNDC5 expression . (a) Graphic representation of the miR-665-3p binding motifs within the 3ʹ-UTR of FNDC5. (b-c) The mRNA and protein levels of FNDC5 in the liver from HFD mice. (d) The levels of hepatic irisin detected by a commercial ELISA kit. (e) Relative luciferase activity of the reporter constructs containing either WT or TRU 3ʹ-UTR of FNDC5 after treatment with miR-665-3p agomir. (f) The mRNA levels of FNDC5 in the liver from HFD mice treated with shFNDC5 or shRNA. (g) The levels of AMPKα phosphorylation. (h) Hepatic hydroxyproline levels. (i) Serum ALT and AST levels. All results were expressed as the means ± standard deviations, n = 6 for each group, and * P < 0.05 was considered statistically significant.

Article Snippet: Irisin ELISA Kit (#SK00170-01) was purchased from Aviscera Bioscience (Santa Clara, CA, USA).

Techniques: Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Construct, shRNA, Phospho-proteomics