Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles

doi: 10.1007/s00018-013-1328-y

Figure Lengend Snippet: EVs from astrocytes are originated from MVBs. a Twelve fractions of isopycnic density centrifugation of CM were collected, separated by SDS-PAGE, and immunoblotted for STI1 (upper panel) and ApoE (lower panel). Densitometry of the bands is shown on the graph along with a density plot of the entire gradient. b After gel filtration chromatography of fractionated CM fractions 1–2, 5–6, 11–12, and 16–17 were immunoblotted for transferrin receptor (TfR), HSP90, HSP70, STI1, flotillin, Tgs101, Vps36, PrPC, and caveolin. c Astrocytes were co-transfected with STI1-GFP and mCherry Rab5 or Rab7 and examined by TIRF microscopy. Scale bar 20 μm. d Astrocytes were co-transfected with STI1–CFP and wild-type VPS4A-EGFP (VPS4–WT–EGFP) or its dominant-negative mutant E228Q (VPS4–Dn–eGFP), and examined by confocal microscopy. e Total number of vesicles in CM was counted by nanoparticle tracking and f levels of STI1 released were measured by ELISA in CM from astrocytes transfected with wild-type pEGFP–VPS4A (VPS4 WT) or its dominant-negative form pEGFP–VPS4A-E228Q (VPS4DN). Data represent mean ± SD of three independent experiments in triplicate. * p < 0.05. g Astrocyte cultures immunostained for VPS4A (red) and STI1 (green) were analyzed by ground-state depletion and individual molecule return (GSDIM) microscopy. Scale bars are 7.5 μm for wide-field and 2.5 μm for GSD super-resolution images

Article Snippet: Microvesicle analysis and quantification Conditioned medium was fractioned by sequential ultracentrifugation or gel filtration chromatography, and the number of particles and particle size was counted by a nanoparticle tracking analysis device (Nanosight LM20, coupled to a CCD camera and a laser emitting a 60-mW beam at 405-nm wavelength).

Techniques: Centrifugation, SDS Page, Filtration, Chromatography, Transfection, Microscopy, Dominant Negative Mutation, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles

doi: 10.1007/s00018-013-1328-y

Figure Lengend Snippet: STI1 and PrPC are present in lipid compartments. a–c CM from astrocytes was untreated (a) or treated with SDS (b) followed by gel filtration chromatography (Superose 12 prep grade, GE Healthcare) and collection of 25 fractions. The proteins were precipitated with TCA and separated by SDS-PAGE, followed by immunoblotting with antibodies for STI1 and PrPC and c quantified by densitometry. d Semi-confluent astrocyte cultures were labeled with [9,10-3H] myristic acid. After 3 days, the cells were washed with PBS and the medium was conditioned for 48 h. The CM was run through gel filtration, lipids were extracted with methanol-chloroform, and the fractions were counted using liquid scintillography. The graph compares the contents of STI1 (absolute values) and lipids (cpm). e The number of vesicles in fractions 1–2, 5–6, and 11–12 were analyzed by nanoparticle tracking. f The number of vesicles in the pellets of 1, 2, or 16 h of centrifugation was analyzed by nanoparticle tracking. g Sizes of vesicles in fractions 1–2, 5–6, and 11–12 measured by particle tracking and compared with the sizes of vesicles in sedimentable fractions from 1, 2, and 16 h of centrifugation

Article Snippet: Microvesicle analysis and quantification Conditioned medium was fractioned by sequential ultracentrifugation or gel filtration chromatography, and the number of particles and particle size was counted by a nanoparticle tracking analysis device (Nanosight LM20, coupled to a CCD camera and a laser emitting a 60-mW beam at 405-nm wavelength).

Techniques: Filtration, Chromatography, SDS Page, Western Blot, Labeling, Centrifugation