Journal: International Journal of Oral Science
Article Title: A blood glucose fluctuation-responsive delivery system promotes bone regeneration and the repair function of Smpd3-reprogrammed BMSC-derived exosomes
doi: 10.1038/s41368-024-00328-6
Figure Lengend Snippet: Smpd3 mediates osteogenic differentiation of BMSCs through autophagy. a After pretreatment with 50 μmol/L GW4869 (Smpd3 inhibitor) for 1 h to inhibit Smpd3 in the presence or absence of CQ. b Treatment with 5 μmol/L GW4869 and Smpd3 knockdown lentivirus inhibited Smpd3 expression, and LC3 puncta were assessed using immunofluorescence analysis. Smpd3 inhibition reduced the formation of LC3 puncta (green dots in the figure). c Overexpression of Smpd3 increased autophagic flux. Smpd3 knockdown downregulated LC3 and upregulated P62, while overexpression of Smpd3 upregulated LC3 and downregulated P62. d Direct overexpression of Smpd3 can induce 50 μmol/L MCQ to inhibit LC3 autophagic flux and increase LC3 puncta. e Smpd3 knockdown inhibited the expression of p-p38 MAPK induced by 1xEBSS (autophagy inducer), while overexpression of Smpd3 activated the expression of p-p38 MAPK induced by 1xEBSS. f Smpd3 knockdown activated 1xEBSS-induced p-p38 MAPK expression, while overexpression of Smpd3 inhibited 1xEBSS-induced p-p38 MAPK expression. g , h After activation of autophagy with 1xEBSS, shSmpd3 increased bone formation at 7 days and ARS staining at 14 days. i , j There was no significant difference in ALP and calcium ion levels between the osteogenic induction group and the siControl group at 3 days and 14 days. k – n After inhibition of autophagy by 50 μmol/L CQ, Smpd3 overexpression increased in ALP staining at 7 days of bone induction and ARS staining at 14 days of bone induction, with significant difference in ALP quantification and calcium ion quantification at 3 days of bone induction compared to those of the vector group. o Smpd3-mediated autophagic induction is achieved by activating p38 MAPK, inhibiting mTOR and promoting osteogenic differentiation of BMSCs by inducing autophagy (scale bar = 50 μm, * P < 0.05, ** P < 0.01, *** P < 0.001, *** *P < 0.000 1 using one-way ANOVA and Student-Newman-Keuls test)
Article Snippet: The membrane was sealed with 5% dehydrated milk in TBS-T (20 mmol/L Tris HCl, pH 7.6, 150 mmol/L NaCl, 0.05% Tween 20) for 1 h and then incubated with the primary antibody at 4 °C for 24 h. The primary antibodies used were anti-Smpd3 (A10197; 1:1 000, ABclonal), anti-Runx2 (A2851; 1:1 000, ABclonal), anti-ALP (ab307726; 1:1 000, Abcam), anti-OCN (ab133612; 1:1 000, Abcam), anti- β-actin (AC038; 1:10 000, ABclonal), anti-P62 (sc-28359; 1:1 000, Santa Cruz), anti-LC3A/LC3B (A5618; 1:1 000, ABclonal), anti-p-p38 MAPK (AP0526,1:1 000, ABclonal), anti-p38 MAPK (A14401; 1:1 000, ABclonal), anti-p-mTOR (AP0115; 1:1 000, ABclonal), anti-mTOR (A2445; 1:1 000, ABclonal), anti-GAPDH (AC002); 1:10 000, ABclonal, anti-CD63 (A19023; 1:1 000, ABclonal), anti-CD9 (A19027; 1:1 000, ABclonal), anti-β- tubulin (AC008; 1:1 000, ABclonal), and anti-histone H3 (A2348; 1:1 000, ABclonal).
Techniques: Knockdown, Expressing, Immunofluorescence, Inhibition, Over Expression, Activation Assay, Staining, Plasmid Preparation
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