Summary of Study ST000898

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000624. The data can be accessed directly via it's Project DOI: 10.21228/M8109M This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST000898
Study TitleTAp73 is a marker of glutamine addiction in medulloblastoma
Study TypesiRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 according to the supplier’s protocol for 48 hours
Study SummaryMetabolically-targeted therapies hold the promise of offering an effective and less toxic treatment for tumours including medulloblastoma, the most common malignant brain tumour of childhood. Current treatment relies on the sensitivity of these tumours to DNA damage that was discovered more than 50 years ago. Finding new tumour-specific susceptibilities to complement sensitivity to DNA damage is key to developing new more effective adjuvant therapies. The specific metabolic program of tumours is an attractive vulnerability, as restriction diet are low cost and easy to implement. Here, we present compelling pre-clinical evidence that glutamine restriction diet can be used as an adjuvant treatment for p73-expressing medulloblastoma.
Institute
Queen Mary University of London
DepartmentBlizard Institute
LaboratoryCentre for Genomics and Child Health
Last NameMarino
First NameSilvia
Address4 Newark Street, E1 2AT, London
Emails.marino@qmul.ac.uk
Phone+44 20 7882 2360
Submit Date2017-08-24
Num Groups2
Total Subjects18
Study CommentsWe include 3 biological replicate with 3 technical replicates for each condition.
Raw Data AvailableYes
Analysis Type DetailLC-MS
Release Date2017-11-20
Release Version1
Silvia Marino Silvia Marino
https://dx.doi.org/10.21228/M8109M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR000624
Project DOI:doi: 10.21228/M8109M
Project Title:TAp73 is a marker of glutamine addiction in medulloblastoma
Project Type:siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 according to the supplier’s protocol for 48 hours
Project Summary:Metabolically-targeted therapies hold the promise of offering an effective and less toxic treatment for tumours including medulloblastoma, the most common malignant brain tumour of childhood. Current treatment relies on the sensitivity of these tumours to DNA damage that was discovered more than 50 years ago. Finding new tumour-specific susceptibilities to complement sensitivity to DNA damage is key to developing new more effective adjuvant therapies. The specific metabolic program of tumours is an attractive vulnerability, as restriction diet are low cost and easy to implement. Here, we present compelling pre-clinical evidence that glutamine restriction diet can be used as an adjuvant treatment for p73-expressing medulloblastoma.
Institute:Queen Mary University of London
Department:Blizard Institute
Laboratory:Centre for Genomics and Child Health
Last Name:Marino
First Name:Silvia
Address:4 Newark Street, E1 2AT, London
Email:s.marino@qmul.ac.uk
Phone:+44 20 7882 2360
Funding Source:Children with Cancer UK fellowship (Reference Nº2014/178)

Subject:

Subject ID:SU000935
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Biosource Or Supplier:ATCC
Cell Strain Details:Daoy (ATCC® HTB-186™)
Subject Comments:cancer cell line
Species Group:Human

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id treatment
SA052758Control_7.1Control
SA052759Control_7.2Control
SA052760Control_6.3Control
SA052761Control_6.2Control
SA052762Control_6.1Control
SA052763Control_8.1Control
SA052764Control_8.3Control
SA052765Control_9.3Control
SA052766Control_1.1Control
SA052767Control_9.2Control
SA052768Control_9.1Control
SA052769Control_5.3Control
SA052770Control_8.2Control
SA052771Control_7.3Control
SA052772Control_2.3Control
SA052773Control_3.1Control
SA052774Control_2.2Control
SA052775Control_2.1Control
SA052776Control_1.2Control
SA052777Control_5.2Control
SA052778Control_3.2Control
SA052779Control_1.3Control
SA052780Control_4.3Control
SA052781Control_3.3Control
SA052782Control_4.2Control
SA052783Control_5.1Control
SA052784Control_4.1Control
SA052785Si p73_7.1Si p73
SA052786Si p73_7.2Si p73
SA052787Si p73_6.1Si p73
SA052788Si p73_6.3Si p73
SA052789Si p73_7.3Si p73
SA052790Si p73_6.2Si p73
SA052791Si p73_9.1Si p73
SA052792Si p73_9.3Si p73
SA052793Si p73_5.3Si p73
SA052794Si p73_9.2Si p73
SA052795Si p73_8.3Si p73
SA052796Si p73_8.2Si p73
SA052797Si p73_8.1Si p73
SA052798Si p73_1.1Si p73
SA052799Si p73_2.2Si p73
SA052800Si p73_2.3Si p73
SA052801Si p73_2.1Si p73
SA052802Si p73_1.3Si p73
SA052803Si p73_1.2Si p73
SA052804Si p73_3.1Si p73
SA052805Si p73_3.2Si p73
SA052806Si p73_4.3Si p73
SA052807Si p73_5.1Si p73
SA052808Si p73_4.2Si p73
SA052809Si p73_4.1Si p73
SA052810Si p73_3.3Si p73
SA052811Si p73_5.2Si p73
Showing results 1 to 54 of 54

Collection:

Collection ID:CO000929
Collection Summary:After 48 hours of transfection the cells were washed three times with ice-cold PBS (3X 5mL) and the cells were collected using a cell scraper.
Sample Type:Cell
Collection Method:Scraper
Tissue Cell Identification:Daoy cell
Tissue Cell Quantity Taken:protein normalization

Treatment:

Treatment ID:TR000949
Treatment Summary:siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 according to the supplier’s protocol for 48 hours
Cell Growth Container:plate
Cell Growth Config:attached
Cell Growth Rate:36 hours
Cell Inoc Proc:lipofectamine 3000
Cell Media:DMEM + GlutaMAX medium (Gibco), supplemented with 10% v/v foetal bovine serum (FBS, Gibco) and penicillin/streptomycin (1 U/ml, Gibco),
Cell Envir Cond:37°C in humidified 5% CO2
Cell Harvesting:After 48 h of transfection the cells were washed three times with ice-cold PBS (3x5 mL) and the celles were collected using a cell scraper. Ice-cold water (3 mL,sterile, MilliQ) was added to the samples. The suspension was transferred to a 15 mL polypropylene tube and then snap-frozen in liquid N2 and stored on ice for 5 min. The cells were lysed by two freeze-thaw cycles where the cells were thawed at 37°C in a water bath and frozen using liquid N2. Subsequently, the cells were sonicated in a
Cell Pct Confluence:80-90%
Cell Media Lastchanged:48 hours

Sample Preparation:

Sampleprep ID:SP000942
Sampleprep Summary:The samples were thawed at room temperature and subjected to centrifugation for 17 min at 3000 rpm and 4°C. A quality control (QC) sample was created by pooling an equal volume from all samples. The aqueous supernatants were transferred to clean extraction tubes followed by addition of chloroform and methanol for the final proportion 2.85:4:4 water:methanol:chloroform. The extraction tubes were gently vortexed and then stored at 8°C for 20 min prior to centrifugation for 20 min at 3000 rpm and 4°C. The aqueous phases were recovered and evaporated to dryness at 40°C under N2. All samples were stored at -80°C after evaporation. Prior to analysis the samples were reconstituted in acetonitrile:Milli-Q water 90:10
Processing Method:lysis, freeze thaw cycles and sonication (see cell harvesting)
Processing Storage Conditions:Room temperature and 8°C during extraction, -80°C before and after extraction
Extraction Method:Liquid Liquid Extraction using CHCl3:MeOH:H2O 4:4:2.85
Extract Storage:Extracts were stored in -80°C after evaporation
Sample Resuspension:Acetonitrile:Milli-Q water 90:10
Cell Type:Daoy cells

Combined analysis:

Analysis ID AN001460 AN001461
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Waters Acquity I-Class Waters Acquity I-Class
Column Waters Acquity BEH Amide (50 x 2.1mm,1.7um) Waters Acquity BEH Amide (50 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Waters Synapt G2 QTOF Waters Synapt G2 QTOF
Ion Mode POSITIVE NEGATIVE
Units Peak Area Peak Area

Chromatography:

Chromatography ID:CH001027
Chromatography Summary:The samples were analyzed on a Acquity UPLC I-class system from Waters. A HILIC-Amide column (1.7 μm, i.d. 2.1x50 mm) from Waters was used for sample separation and a non-linear gradient elution profile from 100 % A to 100 % B was used. Mobile phase A consisted of 90:10 acetonitrile/water with 10 mM ammonium formate and 0.1% FA while mobile phase B consisted of 50:50 acetonitrile/ water with 10 mM ammonium formate and 0.1% FA. The flow rate was 0.3 ml/min, the column temperature 40°C and the injection volume 5 µL.
Instrument Name:Waters Acquity I-Class
Column Name:Waters Acquity BEH Amide (50 x 2.1mm,1.7um)
Column Temperature:40°C
Flow Gradient:Non-linear gradient elution profile from 100 % A to 100 % B was used. In detail; 100% A was kept for 0.5 min then decreased non-linearly (slope-factor 8 in MassLynx) over 12.5 min to 100% B, 100% B was held for 3 min followed by 7 min at 100 % A to re-equilibrate the column for a total run-time of 23 min.
Flow Rate:0.3 ml/min
Injection Temperature:4°C
Sample Injection:5 μl
Solvent A:90% acetonitrile/10% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:50% acetonitrile/50% water; 0.1% formic acid; 10 mM ammonium formate
Analytical Time:23 min total sample run-time
Randomization Order:All samples were analyzed in a randomized order with three QC injections interspaced every eleventh injection
Chromatography Type:HILIC

MS:

MS ID:MS001348
Analysis ID:AN001460
Instrument Name:Waters Synapt G2 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:The raw data was converted to NetCDF files using the software DataBridge (Masslynx version 4.1, Waters). Peak detection and retention time alignment was performed using the R based software XCMS (Smith et al. 2006). The centWave function was used for peak detection and the function parameters were set as follows; the maximal deviation in m/z between scans was set to 8 ppm, the maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal to noise ratio cutoff was set to 10. Retention time correction was performed using the “obiwarp” function.
Ion Mode:POSITIVE
Capillary Temperature:500°C
Capillary Voltage:1 kV
Collision Energy:20- 45 eV collision ramp, MSE acquistion
Source Temperature:120°C
Dataformat:NetCDF
Scan Range Moverz:m/z 50-800
  
MS ID:MS001349
Analysis ID:AN001461
Instrument Name:Waters Synapt G2 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:The raw data was converted to NetCDF files using the software DataBridge (Masslynx version 4.1, Waters). Peak detection and retention time alignment was performed using the R based software XCMS (Smith et al. 2006). The centWave function was used for peak detection and the function parameters were set as follows; the maximal deviation in m/z between scans was set to 8 ppm, the maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal to noise ratio cutoff was set to 10. Retention time correction was performed using the “obiwarp” function.
Ion Mode:NEGATIVE
Capillary Temperature:450°C
Capillary Voltage:2 kV
Collision Energy:20- 45 eV collision ramp, MSE acquistion
Source Temperature:120°C
Dataformat:NetCDF
Scan Range Moverz:m/z 50-800
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