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: Kobe Journal of Medical Sciences

Article Title: Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells

doi:

Figure Lengend Snippet: HEK293 cells transiently transfected with a vector encoding either HA-SphK1 or HA-SphK2 together with control, SphK1-siRNA or SphK2-siRNA were cultured for 72 hr. After cell lysis, the lysates were subjected to SDS-PAGE followed by immunoblot analysis using anti-HA and anti-β-tubulin antibodies (A). K562 cells transfected with control, SphK1-siRNA or SphK2-siRNA were cultured for 48 hr. Cell medium was then changed to exosome-depleted one and cultured for 12 hr. Exosomes were purified from the media and analyzed for cargo contents by immunoblot analysis using anti-TfR1, anti-HSP70, and anti-flotillin 2 antibodies. Cell lysates were also subjected to immunoblot analysis with anti-flotillin 2 antibody (B).

Article Snippet: Anti-EEA1 antibody (catalog number 610456) was purchased from BD Biosciences; anti-TfR1 (catalog number HM2134) from Hycult Biotech; anti-HSP70 (catalog number SPA-815) from Stressgen Biotechnologies; anti-flotillin 2 (catalog number sc-48398) from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Lysis, SDS Page, Western Blot, Purification

Journal: Kobe Journal of Medical Sciences

Article Title: Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells

doi:

Figure Lengend Snippet: K562 cells transiently transfected with a vector encoding either siRNA-resistant HA-SphK2 or HA-SphK2(G248D) together with control or SphK2-siRNA were cultured for 48 hr. Exosomes were prepared as in Fig. 2B and analyzed for cargo contents by immunoblot analysis using anti-flotillin 2 antibody (A). K562 cells were cultured in exosome-depleted medium for 12 hr in the absence or presence of either 10 μM DMS or 50 μM HACPT. Exosomes were purified from the media and analyzed for cargo contents by immunoblot analysis using anti-TfR1, anti-HSP70, and anti-flotillin 2 antibodies (B).

Article Snippet: Anti-EEA1 antibody (catalog number 610456) was purchased from BD Biosciences; anti-TfR1 (catalog number HM2134) from Hycult Biotech; anti-HSP70 (catalog number SPA-815) from Stressgen Biotechnologies; anti-flotillin 2 (catalog number sc-48398) from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Western Blot, Purification

Journal: Journal of Cellular and Molecular Medicine

Article Title: Electroacupuncture Pretreatment Ameliorates Perioperative Neurocognitive Disorder in Aged Mice by Inhibiting Ferroptosis Through the SIRT1 / NRF2 / GPX4 Pathway

doi: 10.1111/jcmm.71021

Figure Lengend Snippet: SIRT1/NRF2/GPX4 pathway is involved in hippocampal ferroptosis in aged mice. (A) WB images and quantification analysis of SIRT1, NRF2 and GPX4 in the hippocampus of aged mice. (B) WB images and quantification analysis of SLC7A11, TFR1, IRP2 and ferritin in the hippocampus of aged mice ( n = 3 per group). (C) qRT‐PCR expression of SIRT1, NRF2, GPX4, SLC7A11, TFR1, IRP2 and ferritin mRNA in the hippocampus of aged mice ( n = 3 per group). Values are presented as mean ± SEM. ** p < 0.01 compared with the C group; # p < 0.05 and ## p < 0.01 compared with the M group; + p < 0.05 and ++ p < 0.01 and compared with the EX group.

Article Snippet: The membrane was then incubated overnight at 4°C with primary antibodies: SIRT1 (1:850; Lot‐19G10A10; BOSTER), NRF2 (1:1500; Cat#YT3189; Immunoway), iron regulatory protein 2 (IRP2) (1:3000; Cat#YN3307; Immunoway), transferrin receptor 1 (TFR1) (1:750; LotNo‐23BP65E1; BOSTER), GPX4 (1:1500; Cat#YN3047; Immunoway), ferritin (1:3000; Cat#YT1692; Immunoway) and SLC7A11 (1:2000; Cat#YT8130; Immunoway).

Techniques: Quantitative RT-PCR, Expressing

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