Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: Colocalization of endocytic BDNF with CD63 or Rab11. ( A ) Representative fluorescence images showing the colocalization of QD-BDNF with CD63- or Rab11-EGFP in astrocytes. QD-BDNF was added for 30–12 min to cultured astrocytes transfected with CD63-EGFP or Rab11-EGFP. Yellow arrowheads indicate QD-BDNF colocalized with the corresponding vesicular markers. Scale bar = 5 μm. ( B ) Bar graphs depict average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). Dotted line: average colocalization ratio between QD-BDNF and MitoTracker (Mito.) as a negative control. *P < 0.05. N = 12–15 cells (194–2,782 QD particles). ( C ) Representative fluorescence images showing the colocalization of QD-BDNF with endogenous CD63 or Rab11 in astrocytes. Yellow arrowheads indicate QD-BDNF colocalized with the corresponding vesicular markers stained with anti-CD63 or anti-Rab11. Scale bar = 5 μm. ( D ) Bar graphs depict average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). Dotted line: average colocalization ratio between QD-BDNF and MitoTracker (Mito.) as a negative control. *P < 0.05. N = 10–16 cells (784-2,110 QD particles).

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Fluorescence, Cell Culture, Transfection, Negative Control, Staining

Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: EV-mediated release of endocytosed BDNF from astrocytes. ( A ) Schematic diagram of the experiment for detecting BDNF-EGFP derived from Neuro2A cells in astrocytic EVs. ( B ) Dot blot showing EGFP expression in cell lysates from nontransfected (non. ; negative control) or BDNF-EGFP-transfected Neuro2a cells. ( C ) Dot blotting was used to detect protein expression in cell lysates and EVs extracted from the media of astrocytes treated with ATP or vehicle after being supplied with media from nontransfected or BDNF-EGFP-transfected Neuro2a cells. Antibodies targeting general markers of the endoplasmic reticulum (ER; an EV-negative marker, calregulin) or EVs (TSG101) were used. ( D ) Representative QD-BDNF kymographs from astrocytes were generated via ImageJ/FIJI and obtained after treatment with vehicle or GW4869 (20 µM) for 1 h prior to time-lapse imaging. Red bar: ATP (100 µM) treatment. Arrow heads: disappearance of QD-BDNF fluorescence. ( E ) Average percentages of ATP-induced QD-BDNF secretion events from vesicles in astrocytes pretreated with either vehicle or GW4869. ****P < 0.0001. N = 9–11 cells (vehicle = 201; GW4869 = 468 QD particles). ( F ) Schematic diagram showing overall process of immunocapture of CD63-positive EVs released from ACM using anti CD63-linked magnetic beads. ( G - H ) Bar graphs depict average concentration of BDNF from ACM using ELISA assay with anti-BDNF (G) or BDNF-EGFP with anti-GFP (H). N = 4 ACM batches derived from different astrocyte cultures. Each dot indicates the result measured from individual batch.

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Derivative Assay, Dot Blot, Expressing, Negative Control, Transfection, Marker, Generated, Imaging, Fluorescence, Magnetic Beads, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: Vamp3-dependent secretion of QD-BDNF from CD63-positive vesicles. ( A ) Representative QD-BDNF kymographs generated from CD63-EGFP-transfected astrocytes. Arrow heads: disappearance of CD63-EGFP-positive QD-BDNF fluorescence. ( B ) Average fractions of secreted QD-BDNF particles with (+) or without CD63 (-) among total secreted QD-BDNF particles. N = 24 cells (232 secreted QD particles). ( C ) Average percentages of ATP-induced QD-BDNF secretion events from vesicles with (+) or without CD63 (-). **P < 0.01, ****P < 0.0001. N = 10–11 cells ( siSCR : 11 cells, 508 QD particles; siVamp3 : 10 cells, 308 QD particles). siSCR : 3 independent experiments with 3–4 cells (107–224 QD particles per experiment); siVamp3 : 3 independent experiments with 3–4 cells (91–114 QD particles per experiment). ( D ) Representative fluorescence images showing the colocalization of QD-BDNF with CD63-EGFP in astrocytes treated with siSCR or siVamp3 . Yellow arrowheads indicate QD-BDNF colocalized with CD63-EGFP. Scale bar = 5 μm. ( E ) Average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). *P < 0.05. N = 12 cells (1,737–1,962 QD particles). ( F ) Representative fluorescence images showing the colocalization of QD-BDNF with endogenous CD63 in astrocytes treated with siSCR or siVamp3 . Yellow arrowheads indicate QD-BDNF colocalized with anti-CD63 signals. Scale bar = 5 μm. ( G ) Average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). *P < 0.05. N = 20–29 cells (2,150-2,464 QD particles).

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Generated, Transfection, Fluorescence

Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: Key resource table.

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Control, Virus, Protease Inhibitor, Plasmid Preparation, Software, Staining, Membrane, Isolation, Enzyme-linked Immunosorbent Assay