Journal: Journal of Extracellular Vesicles

Article Title: Neuronal Apoptotic Bodies Facilitate Japanese Encephalitis Virus Infection and Pathogenicity

doi: 10.1002/jev2.70366

Figure Lengend Snippet: JEV infection induces neuronal apoptosis. (A–C) N2a cells were infected with JEV (MOI = 1) or treated with 20 µM ABT‐263. At 36 and 48 hpi or 18 h post‐treatment, the cells were harvested and subjected to apoptosis assessment using the Caspase‑Glo 3/7 assay (A). Flow cytometric analysis of apoptosis was conducted via Annexin V‐FITC and the viability dye Zombie NIR co‐staining (B). Quantification of the percentages of cells in each quadrant from (B) is shown (C). Q1 (Zombie NIR + /Annexin V − ): naked nuclei; Q2 (Zombie NIR + /Annexin V + ): late apoptotic/necrotic cells; Q3 (Zombie NIR − /Annexin V + ): early apoptotic cells; and Q4 (Zombie NIR − /Annexin V − ): live cells. (D–E) Primary mouse neurons were infected with JEV (MOI = 1) or treated with 5 µM ABT‑263. The cells were harvested at 24, 36 and 48 hpi, or 5 h post‐treatment and subjected to apoptosis detection using the Caspase‑Glo 3/7 assay (D). Immunofluorescence assay was performed using anti‐cleaved caspase‐3 antibody (green) with DAPI counterstaining for nuclei (blue). Scale bar: 100 µm (E). (F–G) Primary neurons and microglia co‐culture were infected with JEV (1 MOI) and harvested at 36 and 48 hpi. The proportions of Annexin V positive neurons and microglia within their respective subpopulations were analyzed by flow cytometry at 36 hpi (left) and 48 hpi (right), respectively (F). The proportions of Annexin V positive neurons and microglia in the total cell population were analyzed by flow cytometry at 36 hpi (left) and 48 hpi (right), respectively (G). (H) Frozen mouse brain sections harvested on day 7 after intraperitoneal challenge were subjected to immunofluorescence staining using an anti‐cleaved caspase‐3 antibody (green) and an anti‐JEV E antibody (red), and the cortical and hippocampal regions were presented. Yellow arrowheads mark JEV/cleaved caspase‐3 double‐positive cells, while white arrowheads denote JEV‐negative, cleaved caspase‐3‐positive cells. Scale bar: 100 µm or 500 µm (Enlarged views). Data are presented as mean ± SD of four biological replicates. Statistical analysis was performed using a two‐tailed unpaired Student's t ‐test (A, C, D and F). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For immunofluorescence (IF) staining, frozen brain sections were rinsed three times with PBS, followed by blocking and permeabilization with 1% BSA containing 0.1% Triton X‐100 for 1 h. Subsequently, sections were incubated overnight at 4°C with primary antibodies, including JEV‐E mAb, ASC rabbit mAb ( A24165 , Abclonal), NLRP3 rabbit pAb (A5652SP, Abclonal), IL‐1β rabbit mAb (A27676, Abclonal), cleaved caspase‐3 (Asp175) rabbit mAb, MAP2 Rabbit mAb (A22205, Abclonal), and Iba1 rabbit mAb, respectively.

Techniques: Infection, Staining, Immunofluorescence, Co-Culture Assay, Flow Cytometry, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Neuronal Apoptotic Bodies Facilitate Japanese Encephalitis Virus Infection and Pathogenicity

doi: 10.1002/jev2.70366

Figure Lengend Snippet: Isolation and characterization of ApoBDs derived from N2a Cells. (A) N2a cells infected with 1 MOI JEV for 48 h were stained with Annexin V‐mCherry and Hoechst 33342, and imaged via confocal microscopy. White arrowheads indicate protrusion vesicles. Scale bar: 10 µm. (B) IEM observation of N2a cells infected with JEV (1 MOI) at 48 hpi. White arrowheads indicate JEV particles (12 nm gold particles); yellow arrowheads indicate cleaved‐caspase 3 (4 nm gold particles). Scale bars: 2 µm (left panel) and 200 nm (right panel). (C) Schematic diagram of ApoBDs isolation. (D) IEM observation of purified ApoBDs. Scale bars: 2 µm (top panels) and 200 nm (bottom panels). (E) Quantification of JEV‑containing vesicles in (D) (n = 24 fields). (F) Flow cytometric assessment of the purity of Annexin V‐FITC‐labeled ApoBDs isolated from JEV‐infected N2a cells, presented as a ridgeline plot. (G) The size distributions of ApoBDs derived from JEV‐infected (JEV‐ApoBDs), STS‐stimulated (STS‐ApoBDs) and ABT‐263‐stimulated (ABT‐263‐ApoBDs) N2a cells were measured using DLS. (H) The protein markers of ApoBDs induced by ABT‐263 and JEV in N2a cells were analyzed by Western blotting. Schematic diagram in (C) was created using BioRender ( https://www.biorender.com ).

Article Snippet: For immunofluorescence (IF) staining, frozen brain sections were rinsed three times with PBS, followed by blocking and permeabilization with 1% BSA containing 0.1% Triton X‐100 for 1 h. Subsequently, sections were incubated overnight at 4°C with primary antibodies, including JEV‐E mAb, ASC rabbit mAb ( A24165 , Abclonal), NLRP3 rabbit pAb (A5652SP, Abclonal), IL‐1β rabbit mAb (A27676, Abclonal), cleaved caspase‐3 (Asp175) rabbit mAb, MAP2 Rabbit mAb (A22205, Abclonal), and Iba1 rabbit mAb, respectively.

Techniques: Isolation, Derivative Assay, Infection, Staining, Confocal Microscopy, Purification, Labeling, Western Blot