tsg101  (Proteintech)

Journal: Journal of Sport and Health Science

Article Title: Long-term aerobic exercise enhances circulating exosomal miR-214-3p to promote endothelial progenitor cell-mediated repair of endothelial damage induced by obesity

doi: 10.1016/j.jshs.2025.101094

Figure Lengend Snippet: Eight weeks of aerobic exercise improved the proliferation and migration capabilities of circulating EPC in both humans and rats with obesity through circulating exosomes. (A) Representative transmission electron microscopy image of exosomes derived from human peripheral blood. Scale bar = 200 nm. (B) Exosome characterization and identification. Exosomes derived from human peripheral blood express TSG101 and CD63. (C) Nanoparticle tracking analysis confirms the presence of exosomes with a peak diameter of 100 nm, characteristic of exosomal size. Quantitative analysis of exosomes derived from human peripheral blood revealed no statistically significant difference in the number of exosomes isolated from equal volumes of circulating blood between the control group and the exercise group ( n = 30 for each group). (D) Cell proliferation assay results showed that exosomes derived from the exercise group significantly enhanced the proliferative capacity of human EPC compared to those from the control group, as measured by the CCK-8 method ( n = 20 for each group). *** p < 0.001, Exercise vs . Control. (E) Scratch assay results showed that exosomes derived from the exercise group significantly promoted the migratory ability of human EPC compared to those from the control group ( n = 5 for each group). * p < 0.05, Exercise vs . Control. (F) Representative images of wound healing in the scratch assay, showcasing the migratory response of human EPC. (G) Characterization of circulating exosomes from rat peripheral blood. (H) Quantitative analysis of exosomes derived from rat peripheral blood revealed no statistically significant difference in the number of exosomes isolated from equal volumes of circulating blood among all groups ( n = 3 for each group). (I) Cell proliferation assays revealed that exosomes derived from the HC group exhibited a diminished capacity to promote EPC proliferation compared to those from the NC group in rats. In contrast, exosomes induced by 8 weeks of aerobic exercise significantly enhanced EPC proliferation ( n : 5–6 for each group). * p < 0.05, HC vs . NC; ## p < 0.01, HE vs . HC. (J) Scratch assays indicated that exosomes derived from the HC group exhibited a diminished capacity to enhance EPC migration rates compared to those from the NC group in rats. In contrast, exosomes induced by 8 weeks of aerobic exercise significantly enhanced EPC migration rates ( n = 4 for each group). ** p < 0.01, HC vs . NC; ## p < 0.01, HE vs . HC. (K) Representative images of wound healing in the scratch assay, showcasing the migratory response of rat EPC. CCK-8 = cell counting kit-8; CD63 = cluster of differentiation 63; EPC = endothelial progenitor cells; HC = the high-fat diet with sedentary group; HE = the high-fat diet with exercise group; NC = the normal diet with sedentary group; TSG101 = tumor susceptibility gene 101.

Article Snippet: The primary antibodies used included PI3K (SC-365290, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), Akt1 (SC-5298, 1:1000; Santa Cruz), p-Akt (Ser473) (66444-1-lg, 1:1000; Proteintech Group, Rosemont, IL, USA), phosphatase and tensin homolog (PTEN) (60300-1-Ig, 1:1000; Proteintech), tumor susceptibility gene 101 (TSG101) (DF8427, 1:1000; Affinity Biosciences, Cincinnati, OH, USA), cluster of differentiation 63 (CD63) (AF5117, 1:1000; Affinity), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GB15002-100, 1:4000; Servicebio).

Techniques: Migration, Transmission Assay, Electron Microscopy, Derivative Assay, Isolation, Control, Proliferation Assay, CCK-8 Assay, Wound Healing Assay, Cell Counting

Journal: Bioactive Materials

Article Title: Intelligent-responsive hydrogel synergistically mediates immune remodel-antibacterial-angiogenesis cascade for diabetic foot ulcer repair

doi: 10.1016/j.bioactmat.2026.03.034

Figure Lengend Snippet: Characterization of DFO@Mn-ZIF-8 and UCMSC-exo. (A) The process of synthesis of Mn-ZIF-8. (B, C) TEM images and elements mapping of DFO@Mn-ZIF-8. (D) Particle diameter of Mn-ZIF-8 and DFO@Mn-ZIF-8. (E) Zeta potential of Mn-ZIF-8 and DFO@Mn-ZIF-8. (F-H) XPS analysis of Mn-ZIF-8. (I, J) XRD analysis of Mn-ZIF-8 and DFO@Mn-ZIF-8. (K) The isolation process of UCMSC-exo. (L) Identification of UCMSC-exo by NTA, and Western blot for CD9, and TSG101. (M) TEM images of UCMSC-exo. Scale bar = 500 nm (left), 50 nm (right). (N) Internalization of UCMSC-exo by RAW 264.7 macrophages after 12 h of incubation. Statistical data are presented as mean ± SD (∗ p < 0.05). ( n = 3).

Article Snippet: Methyl alcohol, Dimethyl sulfoxide (DMSO; General-Reagent, Titan, Shanghai, China); Deferoxamine mesylate (DFO; MCE, New Jersey, USA); Zn(NO 3 ) 2 ·6H 2 O, Mn(NO 3 ) 2 ·4H 2 O, 2-methylimidazole (Aladdin, Shanghai, China); UCMSC-exo (Langfang Kangbao Huitai Biotechnology, Langfang, China); Chitosan (CS; Deacetylation degree ≥95%, Macklin, Shanghai, China), 4-carboxyphenylboronic acid; polyvinyl alcohol (4-CPBA, PVA; Aladdin, Shanghai, China); N-(3-Dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC; Aladdin, Shanghai, China); N-Hydroxysuccinimide (NHS; Aladdin, Shanghai, China); PrimeScript RT Master Mix (Takara, Beijing, China); qPCR SYBR Green Master Mix (Yeasen, Shanghai, China); CD9 antibody (Selleck, USA); TSG101 antibody (Abmart, Shanghai, China); CD86 flow antibody and CD206 flow antibody (BioLegend, USA); IL-6, CD206, HIF-1α, CK14, Ki67, CD31, VEGF and α-SMA (AiFang biological, China).

Techniques: Zeta Potential Analyzer, Isolation, Western Blot, Incubation

Journal: Non-coding RNA Research

Article Title: CircEif3c/miR-96–5p/PHF20L1/MEOX2 axis in perivascular preadipocyte exosomes mediates fibroblast dysfunction and vascular remodeling

doi: 10.1016/j.ncrna.2026.01.006

Figure Lengend Snippet: PVPAC/AF co-culture model confirms that PVPAC-derived exosomes mediated intercellular communication. (A) Schematic diagram of primary PVPAC/AF cells culture with subsequent exosome isolation. (B) PVPAC/AF cells co-culture model. (B1) Schematic of the transwell-based co-culture setup. (B2) Representative TEM micrograph showing exosome morphology, scale bar = 100 nm. (B3) NTA-derived size distribution and concentration profiles of isolated exosomes. (B4) Crystal violet assay assessing cell proliferation under different glucose conditions, scale bar = 200 μm. (B5 and B6) Quantitative histograms corresponding to (B3) and (B4), respectively. Data are compared across mono-vs. co-culture systems under normal (NG) or high glucose (HG). vs NG + AF group, ∗P < 0.05, ∗∗P < 0.01. (C) Confocal microscopy tracking exosome uptake. Scale bar = 50 μm. (C1) PKH67-labeled PVPAC-derived exosomes (green) enriched in PVPAC cytoplasm. (C2) PKH67-labeled AF-derived exosomes abundant within AF cytoplasm. (C3) Time-course imaging displayed PVPAC-Exo accumulation in AFs, peaking at 4 h. (D) Quantification of migration and proliferation capacities in AFs after 24-h treatment with PVPAC-Exo (1 × 10 6 particles/mL), using PBS as a vehicle control, scale bar = 200 μm. (E) Impact of NG, HG, and GW4869 on exosome biology, scale bar = 100 nm. (E1) Morphology assessed by TEM. (E2) Proliferation measured via crystal violet. (E3) Western blot quantification of vimentin and exosomal markers (CD63, TSG101) in AFs. (F) RT-PCR analysis of circEif3c and miR-96–5p in AFs and PVPACs after 24 h NG vs. HG. HG induced highest circEif3c and lowest miR-96–5p expression in PVPACs. (G–K) Systematic comparison of exosomal protein signatures across culture modalities. (G1)Single-cell culture. (G2)Dual-cell co-culture. (G3) Co-culture pre-loaded with 1 × 10 6 /mL PVPAC-Exo. (H–K) Bar graphs present mean ± SD. n (the number of experiments) = 3; one-way ANOVA with Dunnett's post-test. ∗vs. respective NG group: ∗P < 0.05, ∗∗P < 0.01; vs. respective HG group: #P < 0.05, ##P < 0.01.

Article Snippet: Antibodies against MEOX2 (1:1500, #ab262916, Abcam, UK), PHF20L1 (1:1500, #ab118190, Abcam, UK), β-actin (#AC004, 1:5000, ABclone, Wuhan), Bcl-2 (1:2000, #ab182858, Abcam, UK), N-cadherin (1:1500, Abcam, UK), vimentin (1:2000, #ab92547, Abcam, UK), Anti-CD63 (1:1000, #ab315108, Abcam, UK), and TSG101 (1:2000, #28283-1-AP, Proteintech, USA) were purchased.

Techniques: Co-Culture Assay, Derivative Assay, Isolation, Concentration Assay, Crystal Violet Assay, Confocal Microscopy, Labeling, Imaging, Migration, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Comparison, Single Cell

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Preparation and characterization of engineered ADGRG1-targeting and hypoxia-treated EVs. (A)Induced fit docking analysis of ADGRG1-binding peptide (A1TP, 7 peptides) and extracellular domain of ADGRG1 protein (PDB database: 7SF8). (B) Analysis of the binding of the A1TP to purified ADGRG1 proteins using a microscale thermophoresis (MST) binding assay. (C) Induced fit docking analysis of A1TP-PEG and extracellular domain of ADGRG1 protein. (D) The binding free energy between A1TP or A1TP-PEG and ADGRG1 were calculated using molecular dynamics simulations. Lower values indicate more stable interactions, with values less than or equal to −20 considered as stable binding modes. (E) Schematic illustration of the conjugating reaction between DSPE-PEG-Alkyne and A1TP. Schematic illustration of the fabrication of A1TP-HX-EVs through external modification by A1TP anchoring. Specific steps for the synthesis of DSPE-PEG-A1TP (DPA) are shown in . (F) FT-IR analysis showed the characteristic peaks of the DSPE-PEG-A1TP. The new triazole ring itself showed a characteristic C=N stretching vibration, a peak at 1538 cm −1 revealed the successful conjugation of A1TP. (G) H Nuclear magnetic resonance (NMR) spectra of DSPE-PEG-A1TP in D2O. The hydrogen signatures of the phenyl and phenol groups at 7.5-8.0 ppm confirmed the successful conjugation of DSPE to A1TP. (H) Western blot analysis verified the presence of three EV marker proteins (ALIX, TSG101, and CD81) and one EV negative marker (GM130) in EVs, HX-EVs, and A1TP-HX-EVs. (I) Transmission electron microscopy (TEM) images of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 200 nm. (J) Zeta potentials of EVs, HX-EVs and A1TP-HX-EVs, n = 3. Two-tailed unpaired Student's t-test was used for statistical analysis. ns, not significant. A two-tailed unpaired Student's t-test was used for statistical analysis. (K) Representative images of the spherical morphology and dispersion states of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 500 nm. (L) Size distributions of EVs, HX-EVs and A1TP-HX-EVs.

Article Snippet: Finally, the presence of the characteristic EV markers Alix (92880, Cell Signaling Technology), CD81 (56039, Cell Signaling Technology) and TSG101 (sc-7964, Santa Cruz Biotechnology) was confirmed by Western blot analysis.

Techniques: Binding Assay, Purification, Microscale Thermophoresis, Modification, Conjugation Assay, Nuclear Magnetic Resonance, Western Blot, Marker, Transmission Assay, Electron Microscopy, Two Tailed Test, Dispersion

Journal: Bioactive Materials

Article Title: NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties

doi: 10.1016/j.bioactmat.2026.01.006

Figure Lengend Snippet: NsPEFs engineering boosts the production of ADSCs-EVs with superior yield and stability A. Schematic illustration of the high-efficiency extraction of extracellular vesicles (EVs) from adipose-derived stem cells (ADSCs) using nanosecond pulsed electric fields (NsPEFs). B. Representative transmission electron microscopy (TEM) images of isolated Ctrl-ADSCs-EVs and NsPEFs-ADSCs-EVs, showing characteristic cup-shaped morphology and bilayer membrane (scale bars: 150 nm and 75 nm). C. Nanoparticle tracking analysis (NTA) showing the particle size distribution of EVs (n = 3). D. Western blot (WB) analysis confirming the positive expression of EV-specific markers (CD81, CD63, TSG101) and the absence of the negative markers (Calnexin, Histone H3, LaminA/C). Quantification is shown on the right (n = 3). E. The particle concentration of EVs. F. NsPEFs stimulation significantly enhanced both yield and protein output compared to Ctrl-ADSCs-EVs. G. Zeta potential measurement indicating colloidal stability (n = 3). H. Purity assessment expressed as the particle-to-protein ratio ( × 10 9 particles/μg). I. Viability of cells post-NsPEFs-ADSCs-EVs treatment assessed by trypan blue exclusion assay (scale bar: 1.7 mm). Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's post-hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns: not significant.

Article Snippet: The antibodies used and the dilution ratios were as follows:Anti-INOS (1:800, Cohesion), Anti-Arginase 1 (1:800, BOSTER), Anti-LRP6 (1:800, BOSTER), Anti-Beta-catenin (1:800, BOSTER), Anti-CD163 (1:800, Abclonal), Anti-CD86 (1:800, BOSTER), Anti-LGR4 (1:800, Abclonal), Anti-IL-1β (1:800, BOSTER), Anti-IL-10 (1:1000, Bioss), Anti-MMP13 (1:800, BOSTER), Anti-COL2A1 (1:800, BOSTER), Anti-Histone H3 (1:1000, Nature Biosciences), Anti-Lamin A/C (1:1000, Nature Biosciences), Anti-Akt (1:1000, Nature Biosciences), Anti-pAkt (1:1000, Nature Biosciences), Anti-RSPO3 (1:1000, Abcam), Anti-CD63(1:800, BOSTER), Anti-CD81(1:800, BOSTER), Anti-TSG101(1:800, BOSTER), Anti-Calnexin(1:800, BOSTER).

Techniques: Extraction, Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Membrane, Western Blot, Expressing, Concentration Assay, Zeta Potential Analyzer, Trypan Blue Exclusion Assay, Two Tailed Test