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You searched for: EV170047 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV170047 1/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 44%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: GAPDH
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Enzalutamide
EV-producing cells
LNCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected EV-associated proteins
PSA
Not detected contaminants
GAPDH
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution,Mode
Reported size (nm)
120
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170047 3/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 44%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: GAPDH
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
DHT
EV-producing cells
LNCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
0.017
Western Blot
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected EV-associated proteins
PSA
Not detected contaminants
GAPDH
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution,Mode
Reported size (nm)
150
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170047 4/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 44%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: GAPDH
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Charcoal stripped serum
EV-producing cells
LNCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
0.012
Western Blot
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected EV-associated proteins
PSA
Not detected contaminants
GAPDH
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution,Mode
Reported size (nm)
120
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170047 5/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 44%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: GAPDH
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
LNCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
0.005
Western Blot
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected EV-associated proteins
PSA
Not detected contaminants
GAPDH
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution,Mode
Reported size (nm)
150
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170047 6/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 33%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
33% (71st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
DHT
EV-producing cells
DUCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
1.6
Western Blot
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected EV-associated proteins
PSA
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution,Mode
Reported size (nm)
120
EV170047 7/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 33%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
33% (71st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Charcoal stripped serum
EV-producing cells
DUCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
1.2
Western Blot
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected EV-associated proteins
PSA
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution,Mode
Reported size (nm)
120
EV170047 8/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 33%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
33% (71st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
DUCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
1.1
Western Blot
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected EV-associated proteins
PSA
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution,Mode
Reported size (nm)
140
EM
EM-type
Transmission-EM
Image type
Wide-field
EV170047 2/8 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Soekmadji C 2017 0%

Study summary

Full title
All authors
Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC
Journal
Oncotarget
Abstract
Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Enzalutamide
EV-producing cells
DUCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: speed (g)
100000
Wash: time (min)
90
Wash: speed (g)
100000
Protein Concentration Method
Not determined
Fluorescent NTA
Characterization: Particle analysis
TRPS
EM
1 - 8 of 8
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV170047
species
Homo
sapiens
sample type
Cell
culture
cell type
LNCaP
LNCaP
LNCaP
LNCaP
DUCaP
DUCaP
DUCaP
DUCaP
condition
Enzalutamide
DHT
Charcoal
stripped
serum
Control
condition
DHT
Charcoal
stripped
serum
Control
condition
Enzalutamide
isolation protocol
dUC
dUC
dUC
dUC
dUC
dUC
dUC
dUC
case number
1
3
4
5
6
7
8
2
EV-METRIC %
44
44
44
44
33
33
33
0