Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Expression of protease-activated receptors. After thrombin preconditioning (2 U) for 6 h, umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed and subjected to immunoblotting analysis with the indicated antibodies. ( A ) Immunoblot analysis of the expression of protease-activated receptors (PARs) in UCB-MSCs. After thrombin preconditioning, the levels of PARs in the UCB-MSCs were determined by immunoblotting analysis. ( B ) Bar graph showing quantification of the amounts of each PAR. ( C ) Lysates from UCB-MSCs treated with the indicated siRNAs for 24 h were subjected to immunoblot analysis. Cell lysates were analyzed by immunoblotting, and the protein levels were normalized to GAPDH. An asterisk (*) indicates a significant difference vs. naive EVs.

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Derivative Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Protease-activated receptors, PAR1 and PAR3, are involved in extracellular vesicle production and phosphorylation of ERK and AKT in umbilical cord-derived mesenchymal stem cells after thrombin preconditioning. Thrombin-preconditioned umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed for immunoblotting analysis with the indicated antibodies. ( A ) After thrombin treatment and inhibition, changes in the protein levels of endosome markers in the UCB-MSCs were assessed by immunoblotting. ( B ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( C ) After thrombin treatment and inhibition, changes in the protein levels of phosphorylated (p)ERK1/2, p AKT, ERK1/2, and AKT in the UCB-MSCs were assessed by immunoblotting. ( D ) Bar graph showing quantification of p ERK1/2 p AKT, ERK1/2, and AKT. ( E ) After thrombin treatment and inhibition, changes in the levels of endosome markers in the UCB-MSCs were determined by immunoblotting. ( F ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( G ) After thrombin treatment, changes in the expression of p ERK1/2 and p AKT in the UCB-MSCs were determined by immunoblotting. ( H ) Bar graph showing quantification of p ERK1/2 and p AKT levels. ( I ) Bar graph showing quantification of the ratio of p ERK/ERK and p AKT/AKT. Data are presented as mean ± SD. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and, a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSCs ( p < 0.05, two-sample t -test; n = 5 per analysis).

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Inhibition, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Protease-activated receptors, PAR1 and PAR3, are involved in endosome production by umbilical cord-derived mesenchymal stem cells and protein cargo contents in extracellular vesicles after thrombin preconditioning. ( A ) After thrombin preconditioning of umbilical cord-derived mesenchymal stem cells (UCB-MSCs), the number of endosomes was determined by labeling early endosomes with green fluorescent protein(GFP-green) and the nuclei with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI-blue). ( B ) Bar graph showing the quantified intensities of the endosome-GFP signal. ( C ) After treatment with thrombin and thrombin inhibitor, the number of extracellular vesicles (EVs) was determined using a NanoSightNS300. ( D ) The size and number distribution of EVs as measured and analyzed by Nanoparticle Tracking Analysis software. ( E ) After treatment with thrombin and thrombin inhibitor, the levels of VEGF, angiogenin, angiopoietin, and HGF in the EVs were measured by multiplex ELISA. Following treatment with thrombin and thrombin inhibition, EVs were isolated from the conditioned media of UCB-MSC cultures, and VEGF, angiogenin, angiopoietin, and HGF protein levels were assessed. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSC ( p < 0.05, two-sample t -test; n = 6 per analysis).

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Derivative Assay, Labeling, Software, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Isolation