Journal: Journal of Extracellular Vesicles

Article Title: Encapsulating Cas9 into extracellular vesicles by protein myristoylation

doi: 10.1002/jev2.12196

Figure Lengend Snippet: N‐Myristoylation promoted encapsulation of Cas9/sgRNA‐eGFP into EVs. (A) HEK293T‐eGFP cells were transduced with vectors expressing Cas9/sgRNA‐eGFP (a control of non‐myristoylation), mCas9/sgRNA‐eGFP, or mCas9(G2A)/sgRNA‐eGFP (a control of loss myristoylation). The EVs from the above conditioned media were isolated by differential ultracentrifugation. Representative TEM images (top panel) and size distribution (bottom panel) of the isolated EVs were analyzed. (B) HEK293T‐eGFP cells expressing Cas9/sgRNA‐empty (control), Cas9/sgRNA‐eGFP, mCas9/sgRNA‐eGFP, or mCas9(G2A)/sgRNA‐eGFP and their isolated EVs were analyzed for expression levels of Cas9, myristoylated Cas9 (Myr‐Cas9), calnexin (EV negative protein biomarker), and EV positive protein biomarkers syntenin, CD9, and CD63 in total cell lysates (Cells, 20 μg of protein) and total EV lysates (EVs, 2.5 μg of protein) by Western blot. (C) The isolated EVs from the mCas9/sgRNA‐eGFP group were subjected to iodixanol density gradient ultracentrifugation. Twelve gradient fractions were collected. The lysates at iodixanol densities 1.075, 1.077, and 1.106, which contain EVs, were analyzed for levels of syntenin, TSG101, and ANXA2 (biomarkers of EVs), and calreticulin (EV‐negative biomarker) by Western blot. (D) EVs derived from DU145 cells over‐expressing Src kinase (top) or HEK293T cells expressing mCas9/sgRNA‐eGFP (bottom) were treated with/without 0.25% Triton X‐100 and with/without proteinase K (0.5 μg/ml) for 30 min. Expression levels of Src kinase or Cas9, and syntenin (known to be encapsulated into EVs) were determined by Western blot. The data are representative of three experiments. (E) Total RNA was extracted from the cell lysate and EVs in the experimental groups Cas9/sgRNA‐empty (control), Cas9/sgRNA‐eGFP, mCas9/sgRNA‐eGFP, or mCas9(G2A)/sgRNA‐eGFP, and cDNA was synthesized by reverse transcription. The amount of sgRNA (using GAPDH as an internal control) in total cell lysates was determined by real‐time quantitative PCR (RT‐qPCR) using the same amount of RNA (200 ng) from each EV group as template. The amount of sgRNA in EVs of the Cas9/sgRNA‐empty experimental group was set as zero. The relative amount of sgRNA in EVs versus total cell lysate was calculated. (F) Total RNA was isolated from EVs derived from cells expressing mCas9/sgRNA‐eGFP. sgRNA was subjected to reverse transcription and cDNA was PCR amplified and Sanger sequenced. The sgRNA sequence targeting the eGFP gene was confirmed (highlighted in red). The data represent three experiments. (G) EVs derived from cells overexpressing mCas9/sgRNA‐eGFP were treated with/without RNase A (0.5 mg/ml) and with/without 0.25% Triton X‐100 for 20 min. After treatment, total RNA was isolated, and cDNA was synthesized by reverse transcription. The amount of sgRNA‐eGFP was determined by RT‐qPCR and the relative expression of sgRNA‐eGFP was calculated. The amount of sgRNA from the treatment group without RNase A and Triton X‐100 was set as 1. *** p < 0.001 ( n = 3)

Article Snippet: In accordance with the guidelines in MISEV2018 (Théry et al., ), the presence of exosomes in light EVs density fractions were confirmed by immunoblotting using antibodies to exosome markers syntenin‐1 (OriGene #TA347044, clone [3D9‐G9‐H4]; 1:1000), and TSG101 (BD Biosciences #612697, clone [51/TSG101]; 1:500), and the EVs marker ANXA2 (BD #610069, clone [5/Annexin II]; 1:500).

Techniques: Transduction, Expressing, Isolation, Biomarker Assay, Western Blot, Derivative Assay, Synthesized, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Sequencing

Journal: Annals of Translational Medicine

Article Title: Trimethylamine-N-oxide-stimulated hepatocyte-derived exosomes promote inflammation and endothelial dysfunction through nuclear factor-kappa B signaling

doi: 10.21037/atm-21-5043

Figure Lengend Snippet: Isolation and characterization of Exos isolated from hepatocytes culture supernatant. (A) Nanovesicles with diameters of around 100 nm were isolated and purified from the hepatocyte culture supernatant, and visualized under an electron microscope. (B) The size distribution of these nanovesicles were further identified by nanoparticle tracking analysis. (C) The expressions of exosomal identity marker CD81 and TSG101 were detected in the Control-Exos and TMAO-Exos by western blotting. (D) The Exos were labelled with DiI and co-cultured with HAECs for 24 h. The results showed that the DiI-labelled Exos could be taken up by HAECs (400× magnification). Exos, exosomes; CD81, cluster of differentiation 81; TSG101, tumor susceptibility gene 101; TMAO, trimethylamine-N-oxide; HAECs, human aortic endothelial cells.

Article Snippet: CD81 primary antibody was purchased from Servicebio (Wuhan, China), TSG101 was purchased from ZEN BIO (Chengdu, China), nuclear factor-kappa B (NF-κB) p65 and Phospho-NF-κB p65 (p-NF-κB p65) at serine536 (Ser536) were purchased from Cell Signaling Technology (Danvers, MA, USA), and glyceraldehyde phosphate dehydrogenase (GAPDH) was purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Isolation, Purification, Microscopy, Marker, Control, Western Blot, Cell Culture