Journal: Life

Article Title: Protein and Lipid Content of Milk Extracellular Vesicles: A Comparative Overview

doi: 10.3390/life13020401

Figure Lengend Snippet: An overview of studies focused on protein and lipid content of milk EVs from different mammal species. The EV isolation protocols and the analytic methods used in the reviewed studies are reported. The terminology used for milk vesicles is based on the reference cited.

Article Snippet: Grossen P, 2021 , EVs 50–150 nm , Bovine , Sequential ultracentrifugation; OptiPrep TM density gradient , MS-Lipotype GmbH , [ ] .

Techniques: Isolation, Centrifugation, Gradient Centrifugation, Chromatography, Mass Spectrometry, Filtration, Purification, Size-exclusion Chromatography, Two-Dimensional Gel Electrophoresis, Electrophoresis, Thin Layer Chromatography

Journal: Emerging Microbes & Infections

Article Title: Induction of amphotericin B resistance in susceptible Candida auris by extracellular vesicles

doi: 10.1080/22221751.2022.2098058

Figure Lengend Snippet: Differences and similarities of extracellular vesicles (EVs) derived from Candida auris using two culture methods. F3–F8 fractions correspond to densities of 1.044, 1.061, 1.065, 1.087, 1.101, and 1.127 ± 0.01 g/L, respectively. (a) Nanoparticle tracking analysis (NTA) of all separated iodixanol fractions from broth and agar cultures, n = 3. (b) Transmission electron microscopy (TEM) of separated iodixanol fractions from broth and agar cultures. Three grid positions were interrogated per sample, n = 3. Scale bars = 200 nm. (c) Representative TEM micrographs of the fractions containing the highest concentration of EVs. (d) Pooled comparison of the relative amount of EVs found in each fraction measured through NTA and bicinchoninic acid protein measurement assay, n = 3.

Article Snippet: Next, the pellets were resuspended in PBS and then subjected to particle separation via layered iodixanol (OptiPrep, Stemcell Technologies, Canada) density gradient ultracentrifugation at 100,000 ×g for 16 h at 4°C.

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Concentration Assay, Comparison, Bicinchoninic Acid Protein Assay

Journal: Cancer discovery

Article Title: Pharmacological suppression of B7-H4 glycosylation restores antitumor immunity in immune-cold breast cancers

doi: 10.1158/2159-8290.CD-20-0402

Figure Lengend Snippet: (A) SKBR3 cells were treated with 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. Membrane CALR, HSP70 and HSP90 were measured by flow cytometry. (B) MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells were established and treated with 5 μM doxorubicin for 24 h. Immunofluorescence staining of the immunogenic cell death markers CALR on the cell surface was performed. Mean fluorescence index of CALR was quantified by ImageJ. Representative images are shown. (C-D) SKBR3, MDA-MB-468, MDA-MB-468-vector, MDA-MB-468-B7-H4 knockout cells were treated with 1 or 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. p-eIF2a and actin were examined by immunoblotting. Scale bar, 100 μm. (E) Representative paired immunohistochemistry staining of B7-H4 and phospho eIF2α (Ser51) in tissue array BC081120. Statistical analysis of immunohistochemical staining indicates B7-H4 expression is negatively correlated with p-eIF2α expression in breast cancer (r = −0.249, p =8.71x10−3). (F) MDA-MB-468-Flag-hB7-H4 were treated in the presence or absence of doxorubicin (10 μM) and/or NGI-1 (10 μM). Then Flag-hB7-H4 was immunoprecipitated followed by immunoblot. The indicated proteins were examined. (G) Schematic diagram of the procedure of OptiPrep density gradient assay with 24 collected fractions from low to high density is shown. MDA-MB-468-vector and MDA-MB-468-hB7-H4 knockout cells were treated with 10 μM doxorubicin for 24 hr followed by OptiPrep density gradient assay. HSP90, CALR, eIF2α and p-eIF2α in fraction 1 to 13 were examined by immunoblotting. (H) eIF2a was immunoprecipitated in fraction 13 in both MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells followed by immunoblotting. PERK, eIF2α and p-eIF2α were examined.

Article Snippet: OptiPrep density gradient protein fractionation assay To prepare the iodixanol gradient, a 50% (w/v) and 5% (w/v) solution of iodixanol were made by diluting the stock solution (60% (w/v) aqueous iodixanol from StemCell Technologies with 0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl pH7.4.

Techniques: Flow Cytometry, Plasmid Preparation, Knock-Out, Immunofluorescence, Staining, Fluorescence, Western Blot, Immunohistochemistry, Immunohistochemical staining, Expressing, Immunoprecipitation