Journal: Protein & Cell
Article Title: Engineered extracellular vesicles enable high-efficient delivery of intracellular therapeutic proteins
doi: 10.1093/procel/pwae015
Figure Lengend Snippet: IDEA activates the STING signaling pathway through high-efficient intracellular delivery of cGAS. (A–F) Generation and characterization of various engineered EVs from producer cells. (A) Representative images of native EVs, Ctrl EVs, cGAS EVs and gag-cGAS EVs by transmission electron microscopy (TEM) and Cryo-TEM. Scale bar, 50 nm. (B–D) Nanoparticle tracking analyzer determined the size distribution (B), diameter (C) and total number of four types of purified EVs (D). (E) Western blot analysis of Alix, TSG101, CD63, and Calnexin in EVs and corresponding producer cells. (F) Western blot to determine the cGAS expression in producer cells and encapsulation in EVs. (G–J) High-efficient intracellular delivery of cGAS by IDEA activates the STING signaling pathway in vitro . (G) cGAS EVs, but not native EVs or Ctrl EVs, induce STING phase condensation after 4 h of incubation with 293T-STING-mNeonGreen cells. cGAMP was used as positive control. Scale bar, 10 μm. (H) cGAS EVs induce IFN luciferase in THP1-Lucia™ ISG cells. Data are presented as the mean ± SEM, n = 4 biologically independent experiments. (I) Relative IFNβ, CXCL10, TNFα, and IL-6 mRNA levels show STING activation in THP-1 cells. Data are presented as the mean ± SEM, n = 3 biologically independent experiments. (J) THP-1 cells that treated with cGAS EVs exhibited STING activation and TBK-1/IRF-3 phosphorylation. All images in (A) and (G) are representative of at least three biologically independent experiments. Statistical analyses were performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test.
Article Snippet: As described previously , the particle concentration and size distribution of EVs were quantified using a nanoparticle tracking analyzer (Particle Metrix, ZetaView PMX110) equipped with ZetaView 8.04.02 SP2 software according to the manufacturer’s protocol.
Techniques: Transmission Assay, Electron Microscopy, Purification, Western Blot, Expressing, Encapsulation, In Vitro, Incubation, Positive Control, Luciferase, Activation Assay, Phospho-proteomics
