Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: The impact of ligand conjugation on cell uptake of mEVs‐Tf. (a) Schematic illustration of the principle for evaluating the correlation between ligand conjugation and cell uptake. (b) Confocal fluorescence microscopy analysis of the cell uptake of DiD‐labelled blank mEVs and the transferrin conjugated counterparts in the presence or absence of 50 mM free transferrin, respectively. HepG2 cells were incubated with DiD‐labelled (red) mEV‐Tf for 12 h, and stained with Hoechst 33342 (blue) for cell nuclei imaging. (c) Lysosome colocalization analysis of DiD‐labelled blank mEVs and mEVs‐Tf engineered by different methods. HepG2 cells were incubated with DiD‐labelled (red) mEV samples for 24 h, and stained with Hoechst 33342 (blue) and LysoTracker (green) for cell nuclei and lysosome imaging, respectively. (d) Semi‐quantitative analysis of the colocalization degree between mEVs and lysosomes using the Manders' colocalization coefficients M1 and M2. (e) Representative dot‐plots between particle size and ligand number of the mEVs‐Tf‐CF488 engineered by lipid modification (panel‐i), protein modification (panel‐ii) and membrane insertion (panel‐iii), respectively. The transferrin concentration was 50 µM for all three samples. (f), (g) Percentage of Tf‐CF488 positive particles (f) and ligand density (g) of the mEVs‐Tf‐CF488 engineered by different methods with increasing input concentrations of Tf‐CF488. (h) FCM analysis of the impact of ligand density on the cell uptake degree of the DiD‐labelled mEVs‐Tf‐CF488 engineered by lipid modification (panel‐i), protein modification (panel‐ii) and membrane insertion (panel‐iii), respectively. (i,j) The relationship between cell fluorescence intensity (FL‐DiD) and input concentration of Tf‐CF488 (i), and ligand density (j) for DiD‐labelled mEVs‐Tf‐CF488 engineered by different methods.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: Conjugation Assay, Fluorescence, Microscopy, Incubation, Staining, Imaging, Modification, Membrane, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: Impact of ligand conjugation on MFGE8 accessibility and surface‐ligand distribution homogeneity of transferrin. (a) Schematic illustration of ligand conjugation effects on the cell binding behaviour of mEVs‐Tf. (b) FCM analysis of the MFGE8‐mediated cell uptake of mEVs. HepG2 cells were treated with DiD‐labelled blank mEVs with (blue) or without (red) labelling against MFGE8 antibodies. (c) Relationships between particle size and the immunofluorescence intensity of MFGE8 for blank mEVs (panel‐i) and the transferrin‐conjugated mEVs engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (d) The percentage of MFGE8‐positive particles (left y‐axis) and the immunofluorescence intensity (right y‐axis) of the mEV samples. (e) Correlation between the fluorescence intensity of CF488 versus CF594 for individual mEVs‐Tf‐CF488 (panel‐i) and mEVs‐Tf‐CF488&Tf‐CF594 engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (f) The percentage of CF594‐positive particles (left y‐axis) and the relative fluorescence intensity of CF594 to CF488 (right y‐axis) of the mEV samples. Note: In these experiments, the concentrations of sPLD, TCEP and DSPE‐PEG‐MAL for lipid‐, protein‐ and membrane‐based ligand conjugation were 10 U/mL, 2 and 1.25 mM, respectively.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: Conjugation Assay, Binding Assay, Immunofluorescence, Modification, Membrane, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: In vivo cancer targeting performance of ligand‐conjugated mEVs. (a) In vivo fluorescence images of the cancer‐bearing mice injected with DiD‐labelled blank mEVs, and mEVs‐Tf engineered by distinct methods at different time points. (b) Time‐dependent variation in radiant efficiency of the tumour site of the mice treated with various mEV formulations. (c) Ex vivo fluorescence images of tumours and other organs excised from the treated mice at 24 h post‐injection. (d) Radiant efficiency of the tumours and other organs based on the ex vivo fluorescence images. (e) Schematic illustration of the principle for characterizing the IgG and IgM adsorption on mEVs after serum incubation. (f), (g) Representative distributions and the bivariate dot‐plots of the particle size and the immunofluorescence intensity of IgG (f) and IgM (g) of the mouse serum‐incubated blank mEVs (panel‐i), and the transferrin‐conjugated counterparts engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (h)–(j) Immunolabelling ratio (h), immunofluorescence intensity (i), and particle size (j) of the mouse serum‐incubated mEV samples. (k) The ELISA measurement of the accessible transferrin concentration of the mEV samples before and after serum incubation.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: In Vivo, Fluorescence, Injection, Ex Vivo, Adsorption, Incubation, Immunofluorescence, Modification, Membrane, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: bioRxiv

Article Title: Identification of Human Transferrin Receptor as an Entry Co-receptor for Parvovirus B19 Infection of Human Erythroid Progenitor Cells

doi: 10.64898/2026.04.02.715920

Figure Lengend Snippet: (A) Protein diagram. VP1u-APEX2 consists of APEX2 fused to the C-terminus of the unique region of B19V VP1 (VP1u) via a seven-residue glycine-serine linker (GGSGGSG), followed by a Flag tag and a 6 × Histidine (His) tag. APEX2 has a linker-Flag-His tag fused at the C-terminus. (B) Analysis of purified proteins. VP1u-APEX2 and APEX2 proteins were expressed in bacteria and purified. Approximately (∼) 1 µg of each protein was separated by SDS-PAGE, followed by Coomassie blue staining. M, molecular weight marker. (C) Confocal microscopy of VP1u-APEX2 entry. 1 × 10 6 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C for 2 h. The cells were then immunostained with α-Flag to visualize internalized proteins under a Leica STED confocal microscope. Scale bar = 10 μm. Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole). (D) Western blotting of APEX2-biotinylated proteins. 1 × 10 7 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C. After 2 h, APEX2-mediated biotinylation was then performed as described in the Materials and Methods and Figure S1 . Biotinylated host proteins were purified with streptavidin-conjugated magnetic beads. The supernatant was collected as the flow-through (FT), and the beads were further washed several times and eluted as the pull-down (PD). Both FT and PD samples were analyzed by SDS-PAGE and immunoblotting using Alexa Fluor 680-conjugated streptavidin. (E) Analysis of VP1u-APEX2-biotinylated/associated proteins using quantitative mass spectrometry (qMS). Three independent PD samples prepared from VP1u-APEX2 and APEX2 (control) treated cells were analyzed by on-bead digestion and qMS. MS data were processed and analyzed as described in the Materials and Methods. The bubble plot shows protein enrichment (log 2 fold change) in the VP1u-APEX2 group relative to the APEX control, with color indicating subcellular localization based on Gene Ontology (GO) annotation. TFRC denotes human transferrin receptor 1 (hTfR).

Article Snippet: Purified proteins: Recombinant hTfR ECD protein tagged with a His-tag at the C-terminus (#11020-H07H) and recombinant human ferritin heavy chain 1/FTH1 (#13217-HNAE) were purchased from SinoBiological (Paoli, PA).

Techniques: Residue, FLAG-tag, Purification, Bacteria, SDS Page, Staining, Molecular Weight, Marker, Confocal Microscopy, Incubation, Microscopy, Western Blot, Magnetic Beads, Mass Spectrometry, Control, Protein Enrichment

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells.

doi: 10.1016/j.biopha.2024.117723

Figure Lengend Snippet: Fig. 5. In vivo HU treatment of Nos2–/– mice impairs HU inhibition of proliferation in erythroid progenitors. a) Schematic representation of experimental setup: Nos2–/– or wild-type (WT) mice were treated orally with 200 mg/kg HU or drinking water for 2 weeks. WT mice were injected with 20 mg/kg of 1400W twice daily for 3 consecutive days. Mouse erythroid progenitors (mERP) were isolated from bone marrow by immunomagnetic cell separation using anti-CD71-PE and anti- Ter119-FITC antibodies. b) Immunocytochemistry for Nos2 protein in mERP isolated from WT mice treated or not with HU. Quantification of Nos2-positive cells. c) Citrulline concentration in the bone marrow of WT and Nos2–/– mice treated or not with HU. d) Colony formation assay showing the number of late erythroid (CFU-E), early erythroid (BFU-E), or granulocyte/macrophage progenitors (CFU-GM) in the bone marrow of WT or Nos2–/– mice treated or not with HU. e) Immunocytochemistry for Ki67 in mERP cells isolated from WT and Nos2–/– mice treated or not with HU. f) Quantification of Ki67-positive cells. g) Cell cycle distribution by flow cytometry showing the percentage of cells in G0/G1, S, or G2/M phases of the cell cycle. c) n = 3, f) n = 5; mean + SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT.

Article Snippet: Bone marrow cells were flushed out of the femurs and tibias of WT or Nos2–/– mice treated with HU or 1400W and resuspended in 100 μL of buffer (1 % PBS, 0.5 % BSA, 2 mM EDTA) and stained with 7 μL of anti-human CD71-PE REAfinityTM antibody (Miltenyi Biotec, 130–120–809, Bergisch Gladbach, Germany) for 15 min at 4◦C.

Techniques: In Vivo, Inhibition, Injection, Isolation, Immunocytochemistry, Concentration Assay, Colony Assay, Flow Cytometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal Release

doi: 10.1016/j.jcmgh.2024.01.001

Figure Lengend Snippet: HEV pORF1 predominantly localizes to CD63-positive vesicular structures. Analyses of subcellular distribution of pORF1 ectopically expressed in A549/D3 cells. ( A ) Confocal microscopy of double-tagged pORF1; EGFP-mCherry Merge displays the subcellular distribution of EGFP ( green )-mCherry ( red ) fusion protein without insertion of pORF1. C-term, C-terminal mCherry; N-term, N-terminal EGFP. ( B ) Particle analysis of only C-terminus–containing vesicles (C-term) and N- and C-terminus-containing vesicles (C+N-terminus). ∗∗∗ P > .0001; ∼300 particles were analyzed. ( C ) Confocal microscopy of C-terminally mScarlet-tagged pORF1 ( red ) and indirect immunofluorescent stain of different organelle marker proteins ( green ); ERGIC53, ERGIC; GM130, Golgi; EEA1, EEs; Rab11a, REs; LC3B, APs; LAMP2, LYs; CD63, MVBs. ( D ) Particle analysis of pORF1-containing vesicles (pORF1 alone) and pORF1- and marker protein–containing vesicles (pORF1 + marker); between ∼200 and ∼1000 particles were analyzed for each organelle marker. Single channels depicted as greyscale images with headers indicating the channel coloring; zoom refers to areas magnified from white rectangles in merge; white ellipses indicate nucleus; scale bar represents 40 μm. APs, autophagosomes; EEs, early endosomes; ERGIC, ER-Golgi intermediate compartment; Golgi, Golgi apparatus; LYs, lysosomes; MVBs, multivesicular bodies; REs, recycling endosomes. Data are displayed as mean ± standard error of the mean. Microscopy performed on Leica TCS SP8 System with 100× objective (numerical arperture 1.4). Images deconvoluted via lightning algorithm using LAS X Control software.

Article Snippet: Primary antibodies were raised against ERGIC53/LMAN1 (lectin mannose-binding 1; Santa Cruz Biotechnologies, sc-365158), GM130/GOLGA2 (golgin A2; Cell Signaling, 12480S), EEA1 (early endosome antigen 1; Abcam, ab206860), Rab11a (Ras-related protein Rab-11A; ProSci, 33-825), (MAP1)LC3B (microtubule-associated protein 1 light chain 3-beta; MBL International, PM036 MBL), LAMP2/CD107b (lysosomal associated membrane protein 2; BD Biosciences, 555803), and CD63 (cluster of differentiation 63; Abcam, ab59479).

Techniques: Confocal Microscopy, Particle Size Analysis, Staining, Marker, Microscopy, Control, Software