Journal: Journal of Extracellular Vesicles

Article Title: Profiling of Extracellular Vesicles of Non‐Small Cell Lung Cancer Reveals Proteins Associated With Osimertinib Resistance

doi: 10.1002/jev2.70219

Figure Lengend Snippet: Mass spectrometry profiling of extracellular vesicles to reveal putative osimertinib resistance associated networks in mutant EGFR driven non‐small cell lung cancer cells . (A) The size distribution of extracellular vesicles (EVs) isolated from H1975 or H1975/OR cell culture media before and after osimertinib exposure for 48 h are presented with respect to particle size (in nm) and concentration (particles/mL). Average data from three biological replicates is shown. (B) EVs and cell extracts were studied by western blot analysis for expression of CD9, alix and syntenin‐1. Calnexin was used to assess general cellular protein contamination in the EVs samples. Five microgram of protein was loaded from each sample (EVs or cell lysate). (C) The total number of EVs at the time of harvest (48 h post osimertinib treatment) was calculated in three independent biological experiments. One‐way Anova, ∗; p value < 0.05 versus H1975. (D) Volcano plots depicting the mass spectrometry (MS) based protein raw expression values in EVs released from H1975 and H1975/OR cells to cell culture media prior ( top panel ) or post osimertinib ( bottom panel ) treatment. The data are based on the mean of three biological replicates. T ‐test, cutoff: p ≤ 0.05. (E) Protein signatures were filtered out from the MS data using Qlucore software, comparing EVs released from H1975/OR cells or H1975 cells pre‐ and post‐osimertinib treatment. Data are based on three biological replicates. (F) Representation of the obtained StringDB analysis signature of the top proteins found in the EVs from H1975/OR cells presented in (E) .

Article Snippet: Briefly, coverslips were incubated with 1 μg/μL silane‐PEG‐Biotin (Laysian Bio, Inc., AL, USA, cat. #145‐40), blocked with casein and conjugated with biotinylated CD9 (Novus Biologicals, Abingdon, United Kingdom, cat. #NB500‐327B) using a streptavidin linker.

Techniques: Mass Spectrometry, Mutagenesis, Isolation, Cell Culture, Concentration Assay, Western Blot, Expressing, Software

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 and FHRE circulate on extracellular vesicles (EVs) in normal human serum (NHS) and normal mouse serum (MS). (A) Detection of FHR-1 in the EV fraction from NHS (EV NHS ) but not from homozygous FHR-1-deficient (EV ΔFHR-1 ) human serum by western blot analysis. FHR-1 was also absent from the supernatant fractions of both sera. Results shown are representative of three experiments. An uncropped gel is shown in . (B) Tracking of EV NHS and EV ΔFHR-1 by live-cell imaging using CLSM. EVs were stained with anti-FHR-1 antibodies conjugated with Alexa Fluor 647 (red). Bars: 10 µm. (C–F) Size distribution and number of vesicles isolated from EV FHR-1 and EV FHRE determined by DLSM using NanoSight NTA 3.2 software. Graphs in ( C,E ) represent overlays of results from 3 to 4 donors. ( (C) , SEM ± standard error, *p ≤ 0.0255 by unpaired two-tailed t-test, n = 4), and ( (E) , SEM ± standard error, *p ≤ 0.0232 by unpaired two-tailed t-test, n = 3). (G) Tracking by live-cell imaging using CLSM of EV FHR-1 and EV ΔFHR-1 stained with anti-CD9 antibody (vesicle marker) and SYTOX orange (nucleic acid marker), Bars: 10 µm. (H) Size distribution of EVs transporting CD9 and FHR-1 (EV CD9&FHR-1 ). EV FHR-1&CD9 were captured with anti-CD9-coated beads from EV FHR-1 , isolated from 1 mL NHS and analyzed by DLSM using NanoSight NTA 3.2 software. (I) Particle numbers (J) and sizes (K) in fractions obtained by size-exclusion chromatography of EV NHS measured by DLSM. (L) High FHR-1 content in fractions 13–16 determined by ELISA. FHR-1 (red) (M) and FHR-1, and vesicle marker CD9, were in close proximity (red) (N) in atherosclerotic tissues, mainly in blood vessels (stippled lines). Complexes were analyzed by proximity ligation assays using anti-FHR-1 and anti-CD9, and were detected by CLSM. EV FHR-1 carry nucleic acids (orange). Bars = 10 µm.

Article Snippet: FHR-1-transporting EVs were captured using beads coated with monoclonal FHR-1 antibody and were stained with Alexa Fluor 647-labeled anti-human CD9 antibody (1:100; Novus Biological, cat no. NB500-327).

Techniques: Western Blot, Live Cell Imaging, Staining, Isolation, Software, Two Tailed Test, Marker, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Ligation

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Round window membrane extracellular vesicles facilitate inner ear drug delivery

doi: 10.1016/j.jconrel.2025.114153

Figure Lengend Snippet: RWM cells release EVs. (A) A schematic showing the extracellular vesicles (EVs) that are released from cells and can be loaded with proteins, mRNAs, lipids, small molecules, and more. Three groups of vesicle sources are shown schematically: MSC EVs: released by porcine bone marrow stem cells, Epi/Fibro EVs: released by epithelial and fibroblast cells of porcine round window membrane-RWM, and Liposomes. The RWM is the port of entry to the inner ear and consists of an outer epithelial layer, a middle fibroblast layer, and an inner epithelial layer. (B) The nanoparticle tracking shows the size distribution of the nanovesicles released by RWM Epithelial (Epi) and Fibroblast (Fibro) cells, as well as Mesenchymal stem cell (MSC), before and after loading with red fluorescent protein (RFP). The transmission electron microscopy (TEM) micrographs showing all three vesicles before and after loading confirm the integrity of the nanovesicles after loading. (C) The flow cytometry analysis of CD63 antibody at FITC-A channel for Epi, Fibro, and MSC vesicles confirmed the CD63+ nanovesicles. The Ctrl group contains only the secondary antibody. (D) The immunoTEM micrographs of the RWM EVs against gold-conjugated CD9, CD63, and CD81 (exosome markers) confirm exosome identity of EVs derived from RWM Fibroblast Cells via Heat Shock. (E) The western blotting analysis of epithelial and fibroblast EVs isolated by serum deprivation (Epi, Fibro) or heat shock (Epi-HS, Fibro-HS) using CD9, CD63, and CD81 antibodies further confirms the nature of nanovesicles as EVs. PNGase F was used to analyze whether a protein is N-glycosylated and to study the impact of glycosylation on its molecular weight. In PNGase + samples, the band between 50 and 90 KDa disappears, and a new band between 30 and 38 KDa is present, confirming the glycosylation of the CD9, CD63, and CD81 proteins.

Article Snippet: Selected antibodies were CD9 (NB500-327, Novus Biologicals), CD63 (NBP2-42225, Novus Biologicals), CD81 (NB100-65805, Novus Biologicals), and B-Actin (3700, Cell Signaling Technologies).

Techniques: Membrane, Liposomes, Transmission Assay, Electron Microscopy, Flow Cytometry, Derivative Assay, Western Blot, Isolation, Glycoproteomics, Molecular Weight

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Round window membrane extracellular vesicles facilitate inner ear drug delivery

doi: 10.1016/j.jconrel.2025.114153

Figure Lengend Snippet: Loading in RWM EVs leads to higher passage across RWM ex vivo and in vivo in pigs. (A) The schematic of the ex-vivo and in-vivo transport test is shown. For the ex-vivo method, the substances are placed on top of the intact, excised RWM, as previously described , in a transwell chamber (without mesh). For the in-vivo method, substances were delivered via IT injection into the middle ear, as previously described , and the inner ear perilymph (20 μL) was collected 1 h after injection from the RWM via a microcapillary tube. The perilymph was then analyzed via mass spectrometry. (B) The concentration of dexamethasone fluorescein (DexF) after passage across RWM explants in transwell and the permeability (Kp) of RWM explant for DexF are shown when DexF is loaded inside Fibro HSEVs, Lipo, and MSC EVs. The Fibro HSEVs had significantly higher passage ex-vivo than naked DexF (biological replicates n: 3, nested 1-way ANOVA p -value: 0.0019). The Fibro HSEVs significantly enhanced the RWM permeability for DexF ex vivo vs naked DexF (biological replicates n: 3, nested 1-way ANOVA p-value: 0.0380). Lipo and MSC EVs did not significantly enhance the RWM permeability for DexF (biological replicates n:3, nested 1-way ANOVA). A plate reader was used for DexF concentration analysis. (C) ImmunoTEM micrographs of the RWM tissue after the Fibro HS EVs passage show the presence of EVs (gold-conjugated CD9, CD63, and CD81) in the middle layer of RWM, confirming their passage across the epithelial barrier. The B-actin used as a control shows specific staining within the fibroblast cells of the RWM. The top row shows lower magnifications, and the bottom row shows higher magnifications. (D) No difference was observed for the concentration of dexamethasone sodium phosphate (DSP) between DSP alone and DSP-loaded Fibro HSEVs ex vivo and in vivo, or between DSP-loaded Fibro HS EVs ex vivo as compared to Fibro HSEVs in vivo. (biological replicates n: 5, One way ANOVA; p -values: 0.9999, 0.1343, 0.8779 for DSP vs. EVs-DSP ex vivo, DSP vs. EVs-DSP in vivo, and EVs-DSP ex vivo vs. EVs-DSP in vivo, respectively. The permeability of the RWM for DSP significantly increased when DSP is loaded inside Fibro HSEVs, both ex vivo and in vivo (biological replicates n: 5, One way ANOVA; p-values:0.0234 and 0.0265 for DSP vs EVs-DSP in ex vivo and in vivo, respectively). No change was observed for EVs-DSP ex vivo compared to in vivo (biological replicates n: 5, One way ANOVA; p-value 0.0693 for EVs-DSP ex vivo vs in vivo). Mass spectrometry was used for DSP concentration analysis.

Article Snippet: Selected antibodies were CD9 (NB500-327, Novus Biologicals), CD63 (NBP2-42225, Novus Biologicals), CD81 (NB100-65805, Novus Biologicals), and B-Actin (3700, Cell Signaling Technologies).

Techniques: Ex Vivo, In Vivo, Injection, Mass Spectrometry, Concentration Assay, Permeability, Control, Staining