Journal: Aging and Disease
Article Title: Bone Regeneration Enhanced by Quercetin-Capped Selenium Nanoparticles via miR206/Connexin43, WNT, and BMP signaling pathways
doi: 10.14336/AD.2025.0025
Figure Lengend Snippet: Qu-SeNPs stimulate osteogenesis via miR-206/connexin 43 (Cx43) pathway . ( A ) Real-time PCR analysis for the miR-206 expression after treating Qu (1 µg mL -1 ) or Qu-SeNPs (75 ng mL -1 ) to MC3T3-E1 cells for 72 h. The results show a fold increase compared to U6 expression (n=3; *p<0.05, vs . control; Paired t-test, and ## p < 0.01; unpaired t-test). ( B ) Real-time PCR analysis for the miR-206 expression in MC3T3 E-1 cells treated with Qu-SeNPs (75 ng mL -1 ) along with miR-206 mimic or miR-206 NC. Relative expression level is compared to control (n=3; **p<0.01, vs . control; one-way ANOVA with Dunnett’s multiple comparisons test, and # p < 0.05, ## p < 0.01; unpaired t-test). ( C ) ALP activity of MC3T3 E-1 cells treated with Qu-SeNPs (75 ng mL -1 ) along with miR-206 mimic or miR-206 NC (n=3; ***p<0.001 vs. control; one-way ANOVA with Dunnett’s multiple comparisons test, and ### p < 0.001; unpaired t-test). ( D ) Luciferase reporter activity of MC3T3 E-1 cells transfected with wild type Cx43 sequence (Cx43 WT) pGL3 plasmid or mutant Cx43 sequence (Cx43 MT) pGL3 plasmid) and treated with miR-206 mimic (n=3; **p<0.01; 2way ANOVA with Sidak’s multiple comparison). (E & F) Relative mRNA and protein expression of endogenous Cx43 in MC3T3E-1 cells treated with miR-206 mimic or miR-206 inhibitor. miR-206 NC or miR-206 inhibitor-NC were used as control (n=3; *p<0.05, **p<0.01; unpaired t-test). ( G ) ALP activity of MC3T3 E-1 cells transfected with Cx43 siRNA, Qu-SeNPs treated alone or along with miR-206 mimic (n=3; ***p<0.001 vs. control; and ## p < 0.01, ### p < 0.001; one-way ANOVA with Tukey’s multiple comparisons test). ( H ) Representative pictures of Sirius Red S and Alizarin red S staining of MC3T3 E-1 cells treated with Cx43 siRNA and miR-206 mimic in osteogenic medium having Qu-SeNPs for 7 days (n=3; ***p<0.001; one-way ANOVA with Tukey’s multiple comparisons test).
Article Snippet: The protein bands were then transferred to a PVDF membrane, blocked with 5 % skim milk, and incubated overnight with antibodies against β-catenin (Cat. No. 610153, BD Biosciences), p-smad (Cat. No. 9511S, Cell signaling Technology), Connexin43 (Cx43) (Cat. No. 3512S, Cell signaling Technology), and β-actin (Cat. No. sc-47778, Santa Cruz Biotechnology) at 4 °C.
Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Activity Assay, Luciferase, Transfection, Sequencing, Plasmid Preparation, Mutagenesis, Comparison, Staining
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