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: Molecular Nutrition & Food Research

Article Title: Bovine Milk‐Derived Extracellular Vesicles Inhibit Catabolic and Inflammatory Processes in Cartilage from Osteoarthritis Patients

doi: 10.1002/mnfr.202100764

Figure Lengend Snippet: Characterization of commercial milk‐derived extracellular vesicles. Within 2 h, EVs were isolated using ultracentrifugation. A) Particle size distribution of isolated vesicles was determined using a NS300. Data presented is a combination of eight separate isolations, error bars represent mean ± SEM. B) Electron microscopy confirmed spherical morphology and biolayer membrane structure. C) Sucrose density gradient following standard ultracentrifugation‐based isolation shows particles in the range of 1.16–1.20 g mL –1 , which is the described range for exosome‐like vesicles. D) Western blotting confirmed the presence of EV‐markers ALIX, HSP70, CD63, and CD81.

Article Snippet: Antibodies used; CD81 (B‐11), Santa Cruz, sc‐166029; HSP70 (3A3), Santa Cruz, sc‐32239: CD63 (H5C6), BD Pharmingen, 556019; ALIX (1A12), Santa Cruz, sc‐53540.

Techniques: Derivative Assay, Isolation, Electron Microscopy, Western Blot

Journal: Cells

Article Title: Novel GATA1 Variant Causing a Bleeding Phenotype Associated with Combined Platelet α-/δ-Storage Pool Deficiency and Mild Dyserythropoiesis Modified by a SLC4A1 Variant

doi: 10.3390/cells11193071

Figure Lengend Snippet: Platelet α- and δ-granule analysis of the GATA1-T296P-variant-affected index patient, his daughter, and his unaffected wife. ( A ) Transmission electron images of platelets from the index patient and his wife as day control. α-granules are colored in brown, δ-granules are indicated as δ; OCS: surface-connected or open canalicular system, where vacuole compartments are colored in blue. ( B ) Immunoblotting analysis of P-selectin (CD62P), CD63, and GPIbα in lysates of washed platelets from the index patient, his daughter, his wife, and an additional day control. Actin labeling served as protein loading control. ( C , D ) Flow cytometric analysis of ( C ) P-selectin/CD62P and ( D ) CD63 platelet surface expression in response to the GPVI-agonist convulxin of the index patient, his daughter, his wife, and additional controls ( n = 30). ( E ) Flow cytometric analysis of mepacrine uptake in platelet δ-granules (convulxin 0 µg/mL) and convulxin-induced release convulxin of the index patient, his daughter, his wife, and additional controls ( n = 30). ( F ) Quantification of serotonin in lysates of washed platelets from the index patient, his daughter, his wife, and additional controls ( n = 10) by using an ELISA assay.

Article Snippet: Monoclonal anti-human CD42b (clone SZ2, 1:200, Beckman Coulter), anti-human CD62P/P-selectin (clone AK-4, 1:40, non-reduced conditions, BD Biosciences), and anti-human CD63 (clone H5C6, 1:40, non-reduced conditions, BD Biosciences) antibodies as well as polyclonal rabbit anti-human β-actin antibodies (1:3000, Cell Signaling Technology, MA, USA) were used as primary antibodies.

Techniques: Variant Assay, Transmission Assay, Western Blot, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Biology

Article Title: Analysis of extracellular vesicle microRNA profiles reveals distinct blood and lymphatic endothelial cell origins

doi: 10.1002/jex2.134

Figure Lengend Snippet: Characterization of enriched extracellular vesicles. (a) Concentration and (b) size of the P10 (microvesicle fraction), P100 (small EV fraction) and S100 (supernatant) fraction after the differential ultracentrifugation enrichment protocol. Particles per mL or nm derived from NTA measurements (11 positions, three technical replicates) are plotted for LEC (blue, n = 5) and BEC (red, n = 2). (c) Representative size distribution histograms for the P10 and P100 fraction from one LEC and BEC donor. (d) Concentration of lipid membrane dye (CMG) stained EVs determined by FITC‐triggered flowcytometry. EVs/mL are plotted for 14 technical replicates for LEC (blue, n = 5) and BEC (red, n = 2). (e) Box plots of the mean fluorescence intensity (MFI) of EV markers (tetraspanins CD81, CD63), endothelial cell surface marker (CD31) and the isotype control detected for CMG‐positive events for the P10 and P100 fraction. Dashed lines are the average MFI of the isotype controls for LEC ( n = 5) and BEC ( n = 2). (f) Percentage of events detected in the respective size range gate of small EVs (grey, <200 nm), intermediate EVs (middle grey, 200–500 nm) and large EVs (dark grey, >500 nm). Data is derived from 14 technical replicates of CMG‐stained P10 and P100 fraction derived from LEC ( n = 5) and BEC ( n = 2). (g) Representative scatter plots for P10 and P100 fraction derived from one LEC and BEC donor. (h) Representative cryo‐TEM images of EV enriched fractions P10 and P100 from LEC and BEC (Scale Bar = 100 nm).

Article Snippet: Staining of respective antigens was performed by addition 1 μL directly PE‐conjugated antibodies for CD63, CD81 (Clones REA1055 and REA513, Miltenyi Biotec, Bergisch Gladbach, Germany) and CD31 (Clone WM59, BD Pharmingen).

Techniques: Concentration Assay, Derivative Assay, Membrane, Staining, Fluorescence, Marker, Control

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Quantitative analysis of mitotic aberrations in BT549 and MDA-MB-231 cells after exposure to 250 nm and 500 nm reversine (REV). ( B, C ) CD63 and F-Actin immunofluorescence labelling in BT549 ( B ) and MDA-MB-231 ( C ) cells treated with 500 nm reversine for 72 hours, compared to DMSO controls. DAPI was used to label nuclei. ( D, E ) Quantification of CD63 immunofluorescence intensity in BT549 ( D ) and MDA-MB-231 ( E ) cells in presence and absence of CIN phenotypes. *p <0.05; **, p <0.01; ***, p-<0.001; ****, p < 0.0001. RFU is relative fluorescence units, REV is reversine. ( F ) Western blot for EV markers for CD63 and CD81.Calnexin and beta-Actin serve as a loading control for whole lysates and negative control for EVs. ( G ) Concentrations of EVs isolated from BT548 (left panel) and MDA-MB-231 (right panel) cells as determined by nanoparticle tracking analysis.

Article Snippet: After blocking with 3% FBS in PBS for 30 minutes, cells were incubated overnight with a CD63 antibody (H5C6; Novus Biologicals, NBP2-42225) diluted in the blocking buffer.

Techniques: Immunofluorescence, Fluorescence, Western Blot, Control, Negative Control, Isolation

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Representative images (left panel) and quantification (right panel) of cell migration and invasion of MDA-MB-231 cells treated with EFEMP1 KD EVs assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( B ) Representative images (left panel) and quantification (right panel) of cell migration and invasion of MDA-MB-231 cells treated with EFEMP1 OEX EVs assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( C ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 scramble and EFEMP1 KD cells determined by Western blot. ( D ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 control and EFEMP1 OEX cells determined by Western blot. ( E ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from DMSO- or REV-treated BT549 scramble and EFEMP1 KD cells determined by Western blot. ( F ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from MDA-MB-231 scramble and EFEMP1 KD cells determined by Western blot. ( G ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from MDA-MB-231 control and EFEMP1 OEX cells determined by Western blot.

Article Snippet: After blocking with 3% FBS in PBS for 30 minutes, cells were incubated overnight with a CD63 antibody (H5C6; Novus Biologicals, NBP2-42225) diluted in the blocking buffer.

Techniques: Migration, Western Blot, Control

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Correlation analysis of EFEMP1 and STAT1 mRNA expression using DepMap data. ( B ) Correlation analysis of EFEMP1 and STAT1 mRNA expression in DepMap-included breast cancer cell lines. ( C ) qPCR quantification of IL6, IL8, CXCL1, CXCL10, EFEMP1, STAT1, STAT3, CD63 and CYLD in BT549 WT and STAT1 KO cells. Statistical analysis was done using two-sided T-test (N = 3; *p< 0.05, **, p< 0.01, ***p < 0.001, ****p < 0.0001). ( D, E ) Representative images ( D ) and quantification ( E ) of cell migration and invasion of BT549 cells co-cultured with EVs isolated from WT of STAT1 KO BT549 cells, treated with DMSO or REV, assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( F, G ) Representative images ( F ) and quantification ( G ) of cell migration and invasion of BT549 cells co-cultured with EVs isolated from STAT1 KO BT549 cells with or without EFEMP1 overexpression, assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm.

Article Snippet: After blocking with 3% FBS in PBS for 30 minutes, cells were incubated overnight with a CD63 antibody (H5C6; Novus Biologicals, NBP2-42225) diluted in the blocking buffer.

Techniques: Expressing, Migration, Cell Culture, Isolation, Over Expression

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 and STAT KO BT549 cells treated with DMSO or REV determined by Western blot. ( B ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from STAT KO BT549 cells treated with or without EFEMP1 overexpression determined by Western blot.

Article Snippet: After blocking with 3% FBS in PBS for 30 minutes, cells were incubated overnight with a CD63 antibody (H5C6; Novus Biologicals, NBP2-42225) diluted in the blocking buffer.

Techniques: Western Blot, Over Expression

Journal: Biology of reproduction

Article Title: Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

doi: 10.1095/biolreprod.108.075481

Figure Lengend Snippet: FIG. 6. In vivo localization of MIR517B and CD63 in the human placenta. The same section of the full-term human placenta was subjected to ISH for MIR517B (A [red]) and subsequent immunostaining for CD63 (B [green]), a marker of the MVB/exosome. C) The merged image of MIR517B and CD63 signals with DAPI-stained nuclei (blue). Some MIR517B signals are colocalized with CD63-positive compartments (arrows) in the STB. The boundaries of STB and fetal capillaries (#) are indicated by white lines. Intervillous space (*) and villous stroma (VS) are indicated. Bar ¼ 10 lm.

Article Snippet: Some hybridized sections were subsequently immunostained with mouse anti-CD63 monoclonal antibodies (catalog number sc-5275, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA; and catalog number H5C6, 1:400; Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: In Vivo, Immunostaining, Marker, Staining

Journal: Biology of reproduction

Article Title: Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

doi: 10.1095/biolreprod.108.075481

Figure Lengend Snippet: FIG. 7. In vitro localization of MIRLET7B and CD63 in trophoblast cells. (A–C) Endogenous CD63 in BeWo cells. A) Immunostaining of CD63. The cells contain CD63-positive vesicular structures (arrows). B) DAPI-stained nuclei of the same cells. C) The merged image of CD63 and DAPI. D–F) Alexa Fluor 594-labeled MIRLET7B was introduced in BeWo cells together with the expression plasmid of EGFP-CD63 as a marker of the MVB/exosome. D) EFGP- CD63-transfected BeWo cells (green). E) Exogenously introduced MIRLET7B in the same EGFP-CD63-expressing cells (red). F) The merged image of CD63 and MIRLET7B signals. Some MIRLET7B signals are colocalized with CD63-positive compartments (arrows). MIRLET7B-negative CD63 compartments are also indicated (arrowheads). Bar ¼ 10 lm.

Article Snippet: Some hybridized sections were subsequently immunostained with mouse anti-CD63 monoclonal antibodies (catalog number sc-5275, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA; and catalog number H5C6, 1:400; Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: In Vitro, Immunostaining, Staining, Labeling, Expressing, Plasmid Preparation, Marker, Transfection

Journal: Biology of reproduction

Article Title: Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

doi: 10.1095/biolreprod.108.075481

Figure Lengend Snippet: FIG. 8. Extracellular release of miRNAs from trophoblast cells via exosomes. RNAs in exosomes enriched from the conditioned medium of EGFP-CD63-expressing BeWo cells were immunoprecipitated using sepharose 4B beads conjugated with isotype-matched nonimmune IgG (cont) or anti-CD63 (aCD63) antibody. The immunoprecipitates were then subjected to real-time RT-PCR analysis for extracellularly released MIR517A (A) and MIR21 (B). Plus sign (þ) indicates that MIR517A was exogenously introduced in EGFP-CD63-expressing BeWo cells. Saccha- romyces cerevisiae tRNA (sc-tRNA) spiked in each RNA sample was used as a control. A normalized miRNA level (miRNA/sc-tRNA) in the immunoprecipitates with anti-CD63 antibody from EGFP-CD63-express- ing BeWo cells without exogenous MIR517A is assigned a value of 1. Values are the mean 6 SD from three measurements. Significantly high levels of both endogenous and exogenous miRNAs were detected in the exosome-enriched fraction compared with those in the negative controls (*P , 0.001).

Article Snippet: Some hybridized sections were subsequently immunostained with mouse anti-CD63 monoclonal antibodies (catalog number sc-5275, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA; and catalog number H5C6, 1:400; Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: Expressing, Immunoprecipitation, Quantitative RT-PCR, Control

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Effects of eribulin or paclitaxel on CD63 localization in HCC1937 cells. HCC1937 cells were treated with vehicle (VEH), 50 nM eribulin (ERB), or 500 nM paclitaxel (PTX) for 4 h to cause maximal microtubule disruption and then co-immunostained for CD63 (green) and β-tubulin (red). ( A ) Immunofluorescence images are representative of two experiments with equal exposures across conditions. Scale bar = 25 µm. ( B ) Quantification of CD63 regional intensity and ( C ) CD63 spots analysis using Operetta™ high-content imaging. Mean cellular fluorescence for a mean of 432 cells (range: 109–1265) was calculated for n = 9 wells and represents three independent experiments. Significance determined by one-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Anti CD63-AF488 antibodies (NBP2–42225AF488, Novus Biologicals, Littleton, CO, USA) were used at a stock concentration of 0.93 mg/mL.

Techniques: Disruption, Immunofluorescence, Imaging, Fluorescence

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Effects of eribulin and paclitaxel on sEV-associated proteins. MDA-MB-231 and HCC1937 cells were treated for 8 h with vehicle, 25 nM eribulin, or 50 nM paclitaxel and sEVs were isolated from the conditioned media. ( A , B ) Representative immunoblots of indicated proteins in 10 µg of cell lysates and equal volumes of sEVs representing 7.5% (CD63, CD9, GAPDH) or 42% (calnexin, CD81, flotillin-1) of the yield of 1.2 × 10 8 MDA-MB-231 cells ( A ) or 6–8 × 10 7 HCC1937 cells ( B ). ( C – F ) Band intensities for indicated proteins in sEV samples normalized to vehicle control sEVs. Points represent the results of independent experiments and bars represent the mean ± SEM, n = 4–6. Significance determined by one-way ANOVA with Tukey’s post-hoc. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Anti CD63-AF488 antibodies (NBP2–42225AF488, Novus Biologicals, Littleton, CO, USA) were used at a stock concentration of 0.93 mg/mL.

Techniques: Isolation, Western Blot, Control

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Fluorescent nanoparticle tracking analysis of CD63 positive sEVs. MDA-MB-231 ( A – D ) or HCC1937 ( E – H ) cells were treated for 8 h with vehicle, 25 nM eribulin, or 50 nM paclitaxel and sEVs collected from the conditioned media. Samples were labeled with anti-CD63-AF488 fluorescent antibodies to quantify CD63+ sEVs and NTA performed with the ZetaView™ in fluorescent mode to measure CD63+ particles. ( A , E ) Overlay of NTA size distribution vs. concentration graphs for each treatment measuring CD63+ sEVs. ( B , F ) The mean CD63+ particle concentration, ( C , G ) percent CD63+ particles, determined by dividing the CD63+ concentration by the total sEV concentration for each sample, and ( D , H ) median CD63+ sEV diameters for each treatment are shown. Points in the bar graphs represent the results of independent experiments and the bars represent the mean ± SEM, n = 4–6, and significance determined by one-way ANOVA with Tukey’s post-hoc test, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Anti CD63-AF488 antibodies (NBP2–42225AF488, Novus Biologicals, Littleton, CO, USA) were used at a stock concentration of 0.93 mg/mL.

Techniques: Labeling, Concentration Assay

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Effects of vinorelbine on sEVs released by MDA-MB-231 cells. Cells were treated for 8 h with vehicle or 25 nM vinorelbine and sEVs collected. ( A ) Representative immunoblots of indicated proteins in 10 µg of cell lysates and equal volumes of sEVs representing 7.5% (CD63, CD9, GAPDH), or 42% (calnexin, CD81, flotillin-1) of the yield of 1.2 × 10 8 MDA-MB-231 cells. ( B ) sEV CD63 signal intensities from the immunoblots were quantified and normalized to vehicle control sEVs for each experiment. Graphs show the mean ± SEM for the relative levels of CD63. n = 4. ( C , D ) sEV samples were labeled with anti-CD63-AF488 fluorescent antibodies and NTA performed with ZetaView in scatter mode to measure total sEVs and in fluorescent mode to measure only CD63+ sEVs. Graphs show overlays of NTA size distribution curves for each treatment condition in total sEVs ( C ) and CD63+ sEVs ( D ). ( E – G ) The mean particle concentrations in total ( E ) and CD63+ sEVs ( F ) and the percent CD63+ particles ( G ), determined by dividing the CD63+ concentration by the total concentration for each sample. ( H , I ) Median particle diameters for each treatment of total ( H ) and CD63+ sEVs ( I ). Points in the bar graphs represent the results of independent experiments and bars represent the mean of these experiments ± SEM, n = 4, and significance determined by unpaired t -test, * p < 0.05, ** p < 0.01.

Article Snippet: Anti CD63-AF488 antibodies (NBP2–42225AF488, Novus Biologicals, Littleton, CO, USA) were used at a stock concentration of 0.93 mg/mL.

Techniques: Western Blot, Control, Labeling, Concentration Assay

Journal: bioRxiv

Article Title: Single-cell RNA Profiling Identifies Diverse Cellular Responses to EWSR1-FLI1 Down-regulation in Ewing Sarcoma

doi: 10.1101/750539

Figure Lengend Snippet: A , C2 in Dormant 2 populations clusters are characterized by increased transcripts for autophagic genes ( GABARAP, MAP1LC3B, MAP1LC3B2 ) and TGFβ receptors and cyclin-dependent kinase inhibitory genes ( CDKN1A, CDKN2A ). B , The relationship between CD63 marker and dormancy associated gene markers ( TGFBR2 and TGFBR3 ), proliferation markers (MKI67, CDKN2A) and EWSR1 and FLI1 in each subpopulation (Pearson correlation). (C0: Stem-like (blue), C1:Committed to the cell-cycle (Red), C2;Dormant-like (Green), C3;Transitional dormant state (Orange).

Article Snippet: The primary antibodies used were human Cyclin B1-153Eu (Fluidigm; 3153009A), Cyclin D-141Pr (Cell Signaling;2978BF), CD271-149Sm (Fluidigm;3149017B), CD40-142Nd (Fluidigm; 3142010B), CD79a-158Gd (Cell Signaling;13333BF), CD49b-161Dy (Fluidigm; 3161012B), CD49e-160Gd (Fluidigm; 3160015B), CD63-150Nd (Fluidigm; 3150021B), CDKN2A-155Gd (Abcam;Ab186932), CELSR2-165Ho (Abcam; Ab189045), FGFR1-159Tb (Cell Signaling;9740BF), MKI67-147Sm (Cell Signaling; 9449BF), MYC-176Yb (Fluidigm;3176012B), TGFBR2-170Er (Abcam;Ab 78419),TGFB1-163Dy (Fluidigm;3163010B), SOX2-169Tm (Cell Signaling;3579BF).

Techniques: Marker

Journal: bioRxiv

Article Title: Single-cell RNA Profiling Identifies Diverse Cellular Responses to EWSR1-FLI1 Down-regulation in Ewing Sarcoma

doi: 10.1101/750539

Figure Lengend Snippet: A , Enrichment of a fraction of EW8 cells with positive dormancy gene features ( CDKN2A, TGFBR2 ) determined by CyTOF in the Dormant 1 and Dormant 2 populations, showing bimodal kinetics over time. B, (VISNE PLOTS) overview of the shift in the frequency of the positive dormant cells over time (120h). C , Distribution of cells with dormant features (TGFBR2+, CDKN2A+, MK167−) in siControl, Dormant 1 and Dormant 2 populations combined. D , Left , Illustration of the gating strategy for detecting cells with high levels of the membrane marker CD63 that are also expressing dormant features (high TGFRB2 and high CDKN2A). CD63-positive, TGFRB2 High , CDKN2A High represents <1% of the population of exponentially growing EW8 cells. Center, and right panels , visualization of the rare fraction of cells isolated from dissociated CDX-ES1 and CDX-EW8 xenografts with dormant characteristics with ViSNE plots. E, Cells dissociated from ES4, ES1 and EW8 xenografts were FACs sorted for high or low CD63 expression, and their ability to migrate was determined with the chemotaxis assay. (P-values are from unpaired t-test).

Article Snippet: The primary antibodies used were human Cyclin B1-153Eu (Fluidigm; 3153009A), Cyclin D-141Pr (Cell Signaling;2978BF), CD271-149Sm (Fluidigm;3149017B), CD40-142Nd (Fluidigm; 3142010B), CD79a-158Gd (Cell Signaling;13333BF), CD49b-161Dy (Fluidigm; 3161012B), CD49e-160Gd (Fluidigm; 3160015B), CD63-150Nd (Fluidigm; 3150021B), CDKN2A-155Gd (Abcam;Ab186932), CELSR2-165Ho (Abcam; Ab189045), FGFR1-159Tb (Cell Signaling;9740BF), MKI67-147Sm (Cell Signaling; 9449BF), MYC-176Yb (Fluidigm;3176012B), TGFBR2-170Er (Abcam;Ab 78419),TGFB1-163Dy (Fluidigm;3163010B), SOX2-169Tm (Cell Signaling;3579BF).

Techniques: Marker, Expressing, Isolation, Chemotaxis Assay

Journal: bioRxiv

Article Title: Single-cell RNA Profiling Identifies Diverse Cellular Responses to EWSR1-FLI1 Down-regulation in Ewing Sarcoma

doi: 10.1101/750539

Figure Lengend Snippet: A , Fluorescence images of EW8 cells transfected with siEWSR1 or siControl; EW8 Cells were fixed after 48h immunostained and subsequently cells were imaged via fluorescent microscopy to study the induction of the autophagy marker LC3B and lysosomal marker CD63 when EWSR1-FLI1 is suppressed. B , Quantification of FAM134B co-localization presented as Pearson’s correlation coefficient (r); ****P < 0.0001, t-test, n=15 fields (∼150 cells) in the dormant cells shown in A using the colocalization tool in imageJ. C , EW8 cells were treated with siEWSR1 or siControl, before being stained for ER resident protein SEC61B. The expansion of ER is shown following EWSR1-FLI1 down regulation (boxed areas). D , Quantification of cells with expanded ER after masking nuclei. **** P, 0.0001, t-test, error bars indicate s.d., n=150 cells.

Article Snippet: The primary antibodies used were human Cyclin B1-153Eu (Fluidigm; 3153009A), Cyclin D-141Pr (Cell Signaling;2978BF), CD271-149Sm (Fluidigm;3149017B), CD40-142Nd (Fluidigm; 3142010B), CD79a-158Gd (Cell Signaling;13333BF), CD49b-161Dy (Fluidigm; 3161012B), CD49e-160Gd (Fluidigm; 3160015B), CD63-150Nd (Fluidigm; 3150021B), CDKN2A-155Gd (Abcam;Ab186932), CELSR2-165Ho (Abcam; Ab189045), FGFR1-159Tb (Cell Signaling;9740BF), MKI67-147Sm (Cell Signaling; 9449BF), MYC-176Yb (Fluidigm;3176012B), TGFBR2-170Er (Abcam;Ab 78419),TGFB1-163Dy (Fluidigm;3163010B), SOX2-169Tm (Cell Signaling;3579BF).

Techniques: Fluorescence, Transfection, Microscopy, Marker, Staining