Journal: Pharmaceutics

Article Title: Exosomes Derived from BMSCs Treated with CeONPs Ameliorate Radiation-Induced Jaw Bone Injury via miR-21-5p/STAT3 Axis-Mediated Osteogenesis and ROS Scavenging

doi: 10.3390/pharmaceutics18020216

Figure Lengend Snippet: miR-21-5p derived from BMSC-Ce-exos regulates STAT3 (wt: wild-type; mut: mutant; NC: negative control). ( A , B ) Heatmap and volcano plot of miRNA sequencing analysis, with blue indicating downregulated miRNAs and red indicating upregulated ones; ( C ) qRT-PCR analysis of the top five expressed miRNAs (miR-140-3p, miR-21-5p, miR-143-3p, miR-27b-3p, and miR-34c-5p) in BMSC-Ce-exos; ( D ) PPI network analysis of intersecting target genes; ( E ) String analysis of the PPI network interaction results; ( F ) Binding sequence of miR-21-5p in the 3′-UTR of STAT3; ( G ) Luciferase readout from BMSCs co-transfected with wt or mut STAT3 3′-UTR and control mimics or miR-21-5p mimics; ( H ) Protein expression of STAT3 in BMSCs transfected with miR-21-5p mimics, miR-21-5p inhibitor and their NCs; ( I ) mRNA expression levels of STAT3 transfected with miR-21-5p mimics, miR-21-5p inhibitor and their NCs in BMSCs. (ns) p > 0.05; (*) p < 0.05; (**) p < 0.01, and (***) p < 0.001.

Article Snippet: Subsequently, 20 μg of proteins were separated via 4–20% SDS-PAGE at 160 V for 40 min and transferred onto polyvinylidene fluoride (PVDF) membranes at 400 mA for 30 min. Membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies against GAPDH (1:5000, HRP-60004, Proteintech, Chicago, IL, USA), ALP (1:500, ab65834, Abcam, Cambridge, UK), Runx2 (1:1000, GTX00792, GeneTex, Irvine, CA, USA), ColA1 (1:1000, GTX112731, GeneTex), OCN (1:1000, DF12303, Affinity, Cincinnati, OH, USA), TSG101 (1:1000, ab125011, Abcam), CD63 (1:1000, 67605-1-lg, Proteintech), Flotillin-2 (1:1000, 66881-1-lg, Proteintech), STAT3 (1:1000, 10253-2-ap, Proteintech), Nrf2 (1:1000, GB113808 , Servicebio, Wuhan, China), Calnexin (1:1000, ab22595, Abcam), and β-Actin (1:1000, GTX109639, GeneTex).

Techniques: Derivative Assay, Mutagenesis, Negative Control, Sequencing, Quantitative RT-PCR, Binding Assay, Luciferase, Transfection, Control, Expressing

Journal: Pharmaceutics

Article Title: Exosomes Derived from BMSCs Treated with CeONPs Ameliorate Radiation-Induced Jaw Bone Injury via miR-21-5p/STAT3 Axis-Mediated Osteogenesis and ROS Scavenging

doi: 10.3390/pharmaceutics18020216

Figure Lengend Snippet: Exosomal miR-21-5p promotes osteogenesis and reduces the ROS levels by targeting STAT3. ( A ) The red staining indicative of mineralization is most obvious when BMSCs are cotransfected with miR-21-5p mimics and pcDNA-NC, while matrix mineralization is significantly reduced when BMSCs are cotransfected with miR-NC and pcDNA-STAT3; ( B ) Quantitative analysis shows that overexpression of STAT3 prevents the enhancement of osteoblastic differentiation in BMSCs induced by miR-21-5p mimics; ( C ) DHE immunofluorescence shows the ROS levels among different groups; ( D ) Quantitative analysis indicates that overexpression of STAT3 prevents the alleviation of ROS accumulation in BMSCs by miR-21-5p mimics; ( E ) SOD assay shows superoxide dismutase levels among different groups; ( F ) MDA assay shows lipid peroxide levels among different groups; ( G ) Western blotting assays shows that overexpression of STAT3 prevents the upregulation of Runx2, Col1a1, ALP, and OCN protein expression induced by miR-21-5p mimics; ( H ) Western blotting assays shows that overexpression of STAT3 prevents the upregulation of Nrf2 protein expression induced by miR-21-5p mimics. (**) p < 0.01; (****) p < 0.0001.

Article Snippet: Subsequently, 20 μg of proteins were separated via 4–20% SDS-PAGE at 160 V for 40 min and transferred onto polyvinylidene fluoride (PVDF) membranes at 400 mA for 30 min. Membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies against GAPDH (1:5000, HRP-60004, Proteintech, Chicago, IL, USA), ALP (1:500, ab65834, Abcam, Cambridge, UK), Runx2 (1:1000, GTX00792, GeneTex, Irvine, CA, USA), ColA1 (1:1000, GTX112731, GeneTex), OCN (1:1000, DF12303, Affinity, Cincinnati, OH, USA), TSG101 (1:1000, ab125011, Abcam), CD63 (1:1000, 67605-1-lg, Proteintech), Flotillin-2 (1:1000, 66881-1-lg, Proteintech), STAT3 (1:1000, 10253-2-ap, Proteintech), Nrf2 (1:1000, GB113808 , Servicebio, Wuhan, China), Calnexin (1:1000, ab22595, Abcam), and β-Actin (1:1000, GTX109639, GeneTex).

Techniques: Staining, Over Expression, Immunofluorescence, Multiple Displacement Amplification, Western Blot, Expressing