Journal: Theranostics

Article Title: Engineered Dll4-overexpressing osteocyte-derived exosomes enhanced bone regeneration by regulating osteogenesis and angiogenesis

doi: 10.7150/thno.121905

Figure Lengend Snippet: Dll4-Exo regulates osteogenic differentiation via Notch signaling pathway. ST2 cells were co-cultured with GFP-Exo or Dll4-Exo in the presence or absence of DAPT for 3 days, followed by qRT-PCR, ALP staining, and western blot. (A) qRT-PCR of Notch target genes ( Hes1, Hey1, HeyL ). (B) qRT-PCR of osteogenic marker genes ( Alpl, Osx, Col1 ). (C) ALP staining. Scale bar = 100 μm. (D) Western blot of osteogenic proteins (Alp, Osx, Runx2). * p < 0.05 , ** p < 0.01 , *** p < 0.001 vs. GFP-Exo group; # p < 0.05 , ## p < 0.01 , ### p < 0.001 vs. same groups without DAPT.

Article Snippet: After fixation and permeabilization, samples were incubated with primary antibodies against Alp, Osx, collagen I, CD31, ɑSMA, osteocalcin (OCN), osteopontin (OPN) (Abcam, UK) and Hes1 (MedChemExpress, USA) overnight at 4 °C, followed by fluorophore-conjugated secondary antibodies for 2 h at RT.

Techniques: Cell Culture, Quantitative RT-PCR, Staining, Western Blot, Marker

Journal: Theranostics

Article Title: Engineered Dll4-overexpressing osteocyte-derived exosomes enhanced bone regeneration by regulating osteogenesis and angiogenesis

doi: 10.7150/thno.121905

Figure Lengend Snippet: Expression of OCN, OPN, Hes1 and CD31 in fracture samples. (A-B) Immunohistochemical staining of OCN, OPN and Hes1 (day 28). Scale bar = 50 μm. (C) Immunofluorescence staining of α-SMA and CD31 (day 14). Scale bar = 100 μm. (D-F) Quantification of OCN, OPN, Hes1 and CD31 expression. * p < 0.05, ** p < 0.01 , *** p < 0.001 vs. PBS control; # p < 0.05 , ## p < 0.01 , ### p < 0.001 vs. GFP-Exo group.

Article Snippet: After fixation and permeabilization, samples were incubated with primary antibodies against Alp, Osx, collagen I, CD31, ɑSMA, osteocalcin (OCN), osteopontin (OPN) (Abcam, UK) and Hes1 (MedChemExpress, USA) overnight at 4 °C, followed by fluorophore-conjugated secondary antibodies for 2 h at RT.

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Control

Journal: Theranostics

Article Title: Engineered Dll4-overexpressing osteocyte-derived exosomes enhanced bone regeneration by regulating osteogenesis and angiogenesis

doi: 10.7150/thno.121905

Figure Lengend Snippet: miR-23a-5p mediates the osteogenic effects but not the angiogenic effects of Dll4-Exo. (A-B) qRT-PCR of osteogenic marker genes ( Alpl , Runx2 , and Osx ) and Notch signaling genes ( Hes1 , Hey1 ) in ST2 cells transfected with miR-23a-5p mimic (mimic-miR) or inhibitor (inhib-miR) and respective controls (mimic-NC, inhib-NC) in the presence of Dll4-Exo. (C, E) Transwell migration assay of HUVECs transfected with mimic-miR or inhib-miR in the presence of Dll4-Exo: representative images (C) and quantitative analysis (E) of migrated cells. Scale bar = 400 μm. (D, F) qRT-PCR of Notch signaling genes ( Hes1 , Hey1 ) (D) and angiogenesis-related genes ( Hif1α , E-cad , and Vegf ) (F) in HUVECs following miR-23a-5p mimic or inhibitor transfection in the presence of Dll4-Exo. * p < 0.05 , ** p < 0.01 , *** p < 0.001 vs. mimic-NC or inhib-NC group.

Article Snippet: After fixation and permeabilization, samples were incubated with primary antibodies against Alp, Osx, collagen I, CD31, ɑSMA, osteocalcin (OCN), osteopontin (OPN) (Abcam, UK) and Hes1 (MedChemExpress, USA) overnight at 4 °C, followed by fluorophore-conjugated secondary antibodies for 2 h at RT.

Techniques: Quantitative RT-PCR, Marker, Transfection, Inhibition, Transwell Migration Assay