Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Western blot analysis to examine relative amounts of exosome marker proteins, CD63, CD81, Tsg101, and Hsp70, recovered in P100 pellets of apical or basolateral medium of Wnt3a-expressing MDCK (MDCK-3a) cells. Equal amounts of P100 samples prepared from apical or basolateral medium were subjected to Western blotting. As standards, small amounts of cell lysates were also loaded. ( b ) The amounts of Wnt3a in S10 sup and P100 pellet from apical or basolateral medium of MDCK-3a cells, as well as the amount in the cell lysate, were analyzed by Western blotting. To compare the amounts of Wnt3a proteins between apical and basolateral media, an equal volume of S10 (10 μl each for apical or basolateral S10 pool) or equal amount of P100 (1/40 of P100 pellets prepared from 400 ml of apical or basolateral media) samples was loaded. In all MDCK cultures in this study, the same volume of medium was added to both apical and basolateral chambers. ( c–f ) Western blotting analysis for detection of activity and amount of Wnt proteins recovered in S10 sup, S100 sup or P100 pellet prepared from either the apical ( c ) or basolateral side ( e ) of MDCK cells. For monitoring of the Wnt3a activity, the amount of stabilized β-catenin induced by the addition of S10 sup, S100 sup or P100 pellet was quantified in L cells, in which β-catenin is undetectable without the addition of Wnt proteins (control; c,e ). In parallel, the amount of Wnt3a used in this activity assay was analyzed ( c,e ). The amounts of β-catenin and Wnt3a in S10, S100 and P100, shown in ( c ) or ( e ), were quantified using Image J software. The ratios of β-catenin to Wnt3a level are indicated in numerical values relative to that in S10, which was set as 100 ( d,f ). The results shown are the mean ± S.D. from 3 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Western Blot, Marker, Expressing, Activity Assay, Software

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a,b ) The P100 pellet of either apical ( a ) or basolateral ( b ) conditioned medium from Wnt3a-expressing MDCK (MDCK-3a) cells was subjected to 0.25–2 M continuous sucrose density-gradient centrifugation. Equal volumes of the collected fractions were analyzed by Western blotting to detect Wnt3a and several markers of exosomes, including Flotillin2 (Flo2), CD63, and Tsg101. In addition, contamination of the ER in the apical sample was examined by use of Calreticulin antibody. Distribution of Hsp70 in apical P100 fractionations is indicated separately in comparison with that of CD63. The results are representative of 4 independent experiments. ( c ) Immunoprecipitation analysis to detect Wnt3a in CD63-containing vesicles. The P100 pellet prepared from apically secreted conditioned medium was incubated with anti-CD63-beads or IgG isotype-beads, and the complexes on the beads were recovered and examined with anti- Wnt3a and CD63 antibodies. ( d ) Immuno-electron microscopy of Wnt3a-containing exosomes released into apical and basolateral media. Vesicles stained with anti-Wnt3a antibody and gold particle-conjugated second antibody were observed by transmission electron microscopy. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Expressing, Gradient Centrifugation, Western Blot, Comparison, Immunoprecipitation, Incubation, Immuno-Electron Microscopy, Staining, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Detection of Wnt11 (indicated by the closed arrow) in culture supernatant (Culture Sup.) and in P100 pellets. MDCK cells expressing Wnt11 were cultured in transwells, and soluble Wnt11 in the culture medium was concentrated by use of Blue Sepharose. Then, concentrated samples were suspended in distilled water; and P100 pellets were prepared as described for Wnt3a. Arrowheads indicate the position of Wnt11 protein. Upper bands shown in culture sup. are non-specifically cross-reactive. ( b ) Sucrose density-gradient centrifugation analysis of Wnt11. The P100 pellet prepared from the apical side of Wnt11-expressing MDCK cells was further fractionated by sucrose density-gradient as was shown in . Wnt11, Flo2, and CD63 were analyzed by Western blotting. (Full-length and smaller forms of CD63 are indicated by the closed and open arrow, respectively) Results shown are representative of 2 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Expressing, Cell Culture, Gradient Centrifugation, Western Blot

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Western blot analysis of P100 pellets to detect recoveries of Wnt3a mutated at the lipidation site, Ser209, produced by MDCK cells. The amounts of Wnt3a in S10 sup and P100 pellet from conditioned medium, as well as the amount in the cell lysate, from MDCK cells expressing the lipidation site mutant Wnt3a (S209A), in which Ser209 was substituted to Ala, were analyzed by Western blotting. Comparative analysis with samples prepared from wild-type Wnt3a-expressing MDCK cells is shown in . ( b,c ) Sucrose density-gradient centrifugation analysis of the lipidation site mutant. The P100 pellet from either the apical ( b ) or basolateral ( c ) side of MDCK cells expressing the S209A mutant of Wnt3a was subjected to continuous sucrose density-gradient as in . The amounts of Wnt3a, Flotillin2 (Flo2), CD63, and Tsg101 were analyzed by Western blotting. Results shown are representative of 3 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Western Blot, Produced, Expressing, Mutagenesis, Gradient Centrifugation