Journal: Materials Today Bio

Article Title: Engineering exosomes from fibroblast growth factor 1 pre-conditioned adipose-derived stem cells promote ischemic skin flaps survival by activating autophagy

doi: 10.1016/j.mtbio.2024.101314

Figure Lengend Snippet: The expression of FGF1 and the identification and comparison of EXO and FEXO. (A) The volcano plot of different mRNAs of human skin flaps. (B) The transcriptome sequencing analysis of human skin flaps. (C) Representative signaling pathways of genes targeted by significantly differential mRNAs according to GO analysis. (D) The transcriptome sequencing analysis of FGFs family in human skin flaps, and the upregulated FGFs in necrotic area were labeled by red rectangle. (E) qPCR result of FGF1, FGF2, FGF9, FGF12, FGF14, FGF16, FGF18, FGF21, FGFR1, FGFR2, FGFR4,FGF7 and FGF11 in necrotic and survived area of mice skin flaps (n = 5). (F) The western blot images of FGF1 in human skin flaps and mice skin flaps. (G)The transmission electron microscopy image of EXO and FEXO. (H) Uptake of Dil-labeled EXO and FEXO into OGD endothelial cells. (I) The particle distribution of EXO and FEXO by NTA analysis. (J) Particle sizes of EXO and FEXO were analyzed (n = 3). (K) Representative signaling pathways of genes targeted by significantly differential microRNAs according to GO analysis of FEXO versus EXO. (L) Western blot analysis of biomarkers of EXO and FEXO. Extracellular vesicle positive marker CD81 and CD9 and negative control calnexin. The gels have been run under the same experimental conditions, and cropped blots were used here. (M) The density value of the proteins previously described in each group (n = 3). (N) The western blotting of VAMP3 in ADSCs, F-ADSCs, F-ADSCs si-NC , F-ADSCs si-VAMP3 . The gels have been run under the same experimental conditions, and cropped blots were used here. (O) The density value of VAMP3 in each group (n = 3). (P) The number of particles produced by each ADSC was analyzed by NTA in ADSC and F-ADSC groups (n = 5). (Q) The viability of OGD endothelial cells by CCK8 analysis (n = 5). (R) The Elisa results of FGF1 in patients serum (n = 3). ∗ stands for P < 0.05, ∗∗ stands for P < 0.01, ∗∗∗ stands for P < 0.001, ns stands for not significant.

Article Snippet: The primary antibodies are following: VEGF (1:1000, Abcam, ab214424), LC3B (1:1000, Huabio, ET1701-65), p62 (1:1000, Proteintech, 66184-1-Ig), NLRP3 (1:1000, Proteintech, 27458-1-AP), GSDMD-N (1:1000, Affinity, DF13758), Cleaved Caspase1 (1:1000, Affinity, AF4005), GPR137 (1:1000, Proteintech, 11929-1-AP), mTOR (1:1000, Huabio, ET1608-5), P-mTOR (1:1000, Huabio, HA721632), PI3K (1:1000, Huabio, HA721673), P-PI3K (1:1000, Abclonal, AP0427), AKT (1:1000, Huabio, ET1609-51), P-AKT (1; 1000, Huabio, ET1607-73) and VAMP3 (1:1000, Abclonal, A8812), FGF1 (1:1000, Zenbio, R24301), GAPDH (1:50000, proteintech, 60004-1-IG).

Techniques: Expressing, Comparison, Sequencing, Protein-Protein interactions, Labeling, Western Blot, Transmission Assay, Electron Microscopy, Marker, Negative Control, Produced, Enzyme-linked Immunosorbent Assay

Journal: Journal of Nanobiotechnology

Article Title: Activation of the Wnt/β-catenin signalling pathway enhances exosome production by hucMSCs and improves their capability to promote diabetic wound healing

doi: 10.1186/s12951-024-02650-x

Figure Lengend Snippet: CHIR99021 increased exosome secretion by promoting SNAP25-dependent exocytosis. ( A ) Volcano plot showing differential gene expression of hucMSCs in the CHIR99021-treated group versus the control group. ( B ) GO enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. ( C ) KEGG enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. ( D ) Heatmap of gene clustering of hucMSCs transcriptomics in synaptic vesicle cycle between CHIR99021-treated and control groups. ( E ) Western blot analysis of β-catenin, Rab27a, Rab7, SNAP25 in hucMSCs treated with Wnt signaling pathway agonists and inhibitors. ( F ) Co-location of SNAP25 with VAMP3 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. ( G ) Co-location of LAMP2 with CD63 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. ( H ) Western blot analysis of SNAP25 knockdown efficiency. ( I ) After knocking down of SNAP25, hucMSCs were treated with Wnt signaling pathway agonists for 48 h, supernatants were collected, and AChE concentration was analysis by ELISA. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: Primary antibodies against the following target proteins were used: SNAP25 (1:100; Proteintech 60159-1-Ig), VAMP3 (1:100; Proteintech 10702-1-AP), LAMP2 (1:200; ABclonal a22482), CD63 (1:200; Proteintech 25682-1-AP), and Rab7a (1:200; ABclonal a12308).

Techniques: Gene Expression, Control, Western Blot, Immunofluorescence, Knockdown, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: miR-124/VAMP3 is a novel therapeutic target for mitigation of surgical trauma-induced microglial activation

doi: 10.1038/s41392-019-0061-x

Figure Lengend Snippet: VAMP3 is a direct target of miR-124. a Overlapping genes from three cell lines overexpressing miR-124. The datasets from three cell lines (HEK 293T, HCT 116, and neuroblastoma CAD) overexpressing ectopic miR-124 were analyzed with the GEO2R tool, and the top 250 differentially expressed genes were included. b mRNA expression of VAMP3, ITGB1, and SERP1 was detected by RT-PCR assays after LPS stimulation in BV2 microglial cells for the indicated hours; * P < 0.05, compared to unstimulated cells, n = 4. c VAMP3 protein expression 24 h after transfection with miR-124 mimics, miR-124 mimics + inhibitor, or negative control; * P < 0.05, compared to the negative control, n = 3. d mRNA expression of VAMP3 24 h after transfection with miR-124 mimics, miR-124 mimics + miR-124 inhibitor or negative control; * P < 0.05, compared to the negative control, n = 3. e Alignment of potential binding sites for miR-124 in the 3′UTRs of VAMP3 mRNA. Renilla/firefly luciferase activity was measured 24 h after cotransfection of plasmids with miR-124 mimics or control mimics; ** P < 0.01, n = 3

Article Snippet: Following transfer to polyvinylidene difluoride (PVDF) membranes, the membranes were blocked for 2 h in 5% nonfat milk at room temperature and were subsequently incubated with primary antibodies against VAMP3 (1:1000, Cell Signaling Technology, USA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10000, Earthox, USA) overnight at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Binding Assay, Luciferase, Activity Assay, Cotransfection, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: miR-124/VAMP3 is a novel therapeutic target for mitigation of surgical trauma-induced microglial activation

doi: 10.1038/s41392-019-0061-x

Figure Lengend Snippet: Kinetics of miR-124/VAMP3 expression after LPS-induced microglial activation (primary microglia and BV2 cell line). a , b Expression of miR-124 was detected by RT-PCR in both the murine BV2 microglial cell line ( a ) and primary microglia ( b ) after LPS stimulation for the indicated hours; *** P < 0.001, compared to the non-stimulated control group, n = 3. c Protein expression of VAMP3 after LPS stimulation for the indicated hours, and the statistics of VAMP3 expression in BV2 cells and primary microglia are shown in d and e , respectively; * P < 0.05, compared to the non-stimulated counterpart control, n = 3. These data are representative of two independent experiments with similar results

Article Snippet: Following transfer to polyvinylidene difluoride (PVDF) membranes, the membranes were blocked for 2 h in 5% nonfat milk at room temperature and were subsequently incubated with primary antibodies against VAMP3 (1:1000, Cell Signaling Technology, USA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10000, Earthox, USA) overnight at 4 °C.

Techniques: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: miR-124/VAMP3 is a novel therapeutic target for mitigation of surgical trauma-induced microglial activation

doi: 10.1038/s41392-019-0061-x

Figure Lengend Snippet: Targeting miR-124/VAMP3 inhibits cytokine release subsequent to LPS stimulation in vitro. a , b BV2 microglial cells were transfected with miR-124 mimics or control mimics or untransfected (naïve control) for 6 h, followed by 100 ng/ml LPS stimulation overnight. Supernatants were collected for ELISAs for IL-6 ( a ) and TNF-α ( b ); * P < 0.05, ** P < 0.01, compared to naïve control and control mimics, n = 3. c The VAMP3 silencing effect of the siRNAs was detected by performing western blotting to detect VAMP3 protein expression in BV2 microglial cells. siRNA-VAMP3-2 showed the best silencing effect and was used to silence VAMP3 for the following experiments. d , e The release of the proinflammatory cytokines IL-6 and TNF-α was detected by ELISAs after VAMP3 silencing; * P < 0.05, compared to the non-silenced control group (si-Con), n = 3. Data are from one experiment representing two independent experiments

Article Snippet: Following transfer to polyvinylidene difluoride (PVDF) membranes, the membranes were blocked for 2 h in 5% nonfat milk at room temperature and were subsequently incubated with primary antibodies against VAMP3 (1:1000, Cell Signaling Technology, USA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10000, Earthox, USA) overnight at 4 °C.

Techniques: In Vitro, Transfection, Control, Western Blot, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: miR-124/VAMP3 is a novel therapeutic target for mitigation of surgical trauma-induced microglial activation

doi: 10.1038/s41392-019-0061-x

Figure Lengend Snippet: The protein expression of VAMP3 is increased after surgery and is modulated by EA and exogenous miR-124 administration. a , b VAMP3 protein expression in the hypothalamus was detected by western blots. c , d VAMP3 protein expression in the hippocampus. * P < 0.05, ** P < 0.01, compared to the control group; # P < 0.05, ## P < 0.01, compared to the surgery group; n = 6. Data are representative of one experiment from three independent experiments

Article Snippet: Following transfer to polyvinylidene difluoride (PVDF) membranes, the membranes were blocked for 2 h in 5% nonfat milk at room temperature and were subsequently incubated with primary antibodies against VAMP3 (1:1000, Cell Signaling Technology, USA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10000, Earthox, USA) overnight at 4 °C.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Nanobiotechnology

Article Title: Activation of the Wnt/β-catenin signalling pathway enhances exosome production by hucMSCs and improves their capability to promote diabetic wound healing

doi: 10.1186/s12951-024-02650-x

Figure Lengend Snippet: CHIR99021 increased exosome secretion by promoting SNAP25-dependent exocytosis. ( A ) Volcano plot showing differential gene expression of hucMSCs in the CHIR99021-treated group versus the control group. ( B ) GO enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. ( C ) KEGG enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. ( D ) Heatmap of gene clustering of hucMSCs transcriptomics in synaptic vesicle cycle between CHIR99021-treated and control groups. ( E ) Western blot analysis of β-catenin, Rab27a, Rab7, SNAP25 in hucMSCs treated with Wnt signaling pathway agonists and inhibitors. ( F ) Co-location of SNAP25 with VAMP3 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. ( G ) Co-location of LAMP2 with CD63 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. ( H ) Western blot analysis of SNAP25 knockdown efficiency. ( I ) After knocking down of SNAP25, hucMSCs were treated with Wnt signaling pathway agonists for 48 h, supernatants were collected, and AChE concentration was analysis by ELISA. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: Primary antibodies against the following target proteins were used: SNAP25 (1:100; Proteintech 60159-1-Ig), VAMP3 (1:100; Proteintech 10702-1-AP), LAMP2 (1:200; ABclonal a22482), CD63 (1:200; Proteintech 25682-1-AP), and Rab7a (1:200; ABclonal a12308).

Techniques: Gene Expression, Control, Western Blot, Immunofluorescence, Knockdown, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Nanobiotechnology

Article Title: Activation of the Wnt/β-catenin signalling pathway enhances exosome production by hucMSCs and improves their capability to promote diabetic wound healing

doi: 10.1186/s12951-024-02650-x

Figure Lengend Snippet: CHIR99021 increased exosome secretion by promoting SNAP25-dependent exocytosis. ( A ) Volcano plot showing differential gene expression of hucMSCs in the CHIR99021-treated group versus the control group. ( B ) GO enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. ( C ) KEGG enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. ( D ) Heatmap of gene clustering of hucMSCs transcriptomics in synaptic vesicle cycle between CHIR99021-treated and control groups. ( E ) Western blot analysis of β-catenin, Rab27a, Rab7, SNAP25 in hucMSCs treated with Wnt signaling pathway agonists and inhibitors. ( F ) Co-location of SNAP25 with VAMP3 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. ( G ) Co-location of LAMP2 with CD63 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. ( H ) Western blot analysis of SNAP25 knockdown efficiency. ( I ) After knocking down of SNAP25, hucMSCs were treated with Wnt signaling pathway agonists for 48 h, supernatants were collected, and AChE concentration was analysis by ELISA. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: Primary antibodies against the following target proteins were used: SNAP25 (1:100; Proteintech 60159-1-Ig), VAMP3 (1:100; Proteintech 10702-1-AP), LAMP2 (1:200; ABclonal a22482), CD63 (1:200; Proteintech 25682-1-AP), and Rab7a (1:200; ABclonal a12308).

Techniques: Gene Expression, Control, Western Blot, Immunofluorescence, Knockdown, Concentration Assay, Enzyme-linked Immunosorbent Assay