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: Molecular & Cellular Proteomics : MCP

Article Title: Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide

doi: 10.1016/j.mcpro.2026.101610

Figure Lengend Snippet: Experimental workflow overview. This schematic illustrates the experimental design used to profile extracellular vesicles (EVs) derived from U87 glioblastoma cells cultured under 2D monolayer and eroid conditions. A , expansion of U-87 MG glioblastoma cells in T225 flasks prior to experimental setup. B , 2D experimental arm: cells cultured in T75 flasks and treated with DMSO, temozolomide (TMZ) 100 μM, or TMZ 200 μM, followed by EV isolation and cell harvesting. C , 3D-spheroid experimental arm: cells seeded in ultra-low attachment (ULA) plates and treated as in ( B ), with subsequent EV isolation and spheroid harvesting. D , EV and cell characterization by Annexin V/PI cell death assay, fluorescence microscopy, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), silver-stained SDS-PAGE, western blotting, and MACSPlex EV flow cytometry assay. E , protein extraction from EVs and cells, followed by proteomic profiling using nano-liquid chromatography tandem mass spectrometry (nLC-ESI-MS/MS). F , differential expressions and functional analysis, including heatmap clustering and identification of GBM-associated protein families (e.g., methyltransferases). G , cross-referencing of selected proteins with The Cancer Genome Atlas (TCGA) for prognostic relevance. H , EV isolation pipeline combining ultrafiltration and size-exclusion chromatography (SEC) for purification of vesicles from conditioned media.

Article Snippet: EV samples from 2D and 3D cultures were analyzed with the MACSPlex EV Kit IO (for immuno-oncology) (#130-108-813) (Miltenyi Biotec).

Techniques: Derivative Assay, Cell Culture, Isolation, Cell Harvesting, Cell Characterization, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy, Staining, SDS Page, Western Blot, Flow Cytometry, Protein Extraction, Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Functional Assay, Size-exclusion Chromatography, Purification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide

doi: 10.1016/j.mcpro.2026.101610

Figure Lengend Snippet: Characterization of EV protein profiles from U87 2D and 3D cultures. A , multiplex flow cytometry using the MACSPlex EV kit was used to profile surface markers of EVs derived from U87 cells cultured under 2D and 3D conditions. CD44 was consistently detected and enriched across conditions, particularly in TMZ-treated samples. The integrins CD29 and CD49e—subunits of the α5β1 integrin heterodimer—were robustly expressed in both 2D and 3D EVs, highlighting their potential role in EV-mediated adhesion and signaling. B , silver-stained SDS-PAGE showing global protein profiles of cell lysates and EVs from 2D and 3D cultures. Distinct banding patterns reflect culture-specific proteomic signatures and successful protein isolation. C , Western blot analysis confirms the presence of established EV-associated proteins, including CD81 and CD44. GAPDH, were used as controls and were detected predominantly in cell lysates, supporting the purity of the EV preparations and absence of major cellular contamination.

Article Snippet: EV samples from 2D and 3D cultures were analyzed with the MACSPlex EV Kit IO (for immuno-oncology) (#130-108-813) (Miltenyi Biotec).

Techniques: Multiplex Assay, Flow Cytometry, Derivative Assay, Cell Culture, Staining, SDS Page, Isolation, Western Blot