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: The primers in qRT-PCR

Article Snippet: The membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam).

Techniques: Sequencing

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: 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

Article Snippet: The membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam).

Techniques: Expressing, Staining, Cell Culture, Quantitative RT-PCR, Western Blot, Gene Expression

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 circ_AFF4 suppresses BM-MSC osteogenic differentiation ( A ) BM-MSCs were transfected with shcirc_AFF4_1–4# or shNC and the expression level of circ_AFF4 in shcirc_AFF4 or shNC transfected BM-MSCs was detected using RT-qPCR. BM-MSCs that were transfected with shcirc_AFF4 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 markers, ALP, OPN, RUNX2 and Colla1 were detected by ( D ) RT-qPCR and ( E ) western blot. * p < 0.05, ** p < 0.001 and *** p < 0.001. Each experiment was performed at least three times independently

Article Snippet: The membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam).

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

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 membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam).

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 membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam).

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