Summary of Study ST003925

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 PR002458. The data can be accessed directly via it's Project DOI: 10.21228/M8X84J This work is supported by NIH grant, U2C- DK119886.

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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 IDST003925
Study TitleAssessing the impact of SLC7A11 silencing on oxidative stress in amoeboid disseminating cancer cells
Study SummaryMetabolomic analysis was performed to investigate changes in oxidative stress status in amoeboid cells. Oxidative stress limits metastasis and amoeboid cancer cells have been identified in a variety of cancers as a subset of metastatic cancer cells characterised by a high Rho-ROCK driven Myosin II activity. Amoeboid migrating cells require lower mitochondrial metabolism, while how they maintain low oxidative stress remains unclear. To interrogate the importance of SLC7A11 in oxidative stress regulation in amoeboid cells, two compounds (menadione and buthionine sulfoximine (BSO)) or an siRNA targeting SLC7A11 (siSLC7A11) were used. To assess the efficacy of these treatments, measurement of oxidised to reduced glutathione ratio (GSH/GSSG), oxidised to reduced nicotinamide adenine dinucleotide (NAD+/NADH) and cysteine levels using LC-MS was performed. Results indicate that treatment timings and concentration with menadione and BSO used throughout the study significantly reduce GSH/GSSG and NAD+/NADH ratio. Additionally, the effect of glutathione supplementation was tested in combination with siSLC7A11. Results indicate that SLC7A11 does not interfere with internalisation of glutathione supplemented in the media as the percentage of labelled glutathione was similar between siSLC7A11 and the siRNA control.
Institute
The Institute of Cancer Research London
DepartmentCell and Molecular Biology
LaboratorySignalling and Cancer Metabolism
Last NamePoulogiannis
First NameGeorge
Address237 Fulham Road SW3 6JB LONDON
Emailgeorge.poulogiannis@icr.ac.uk
Phone+442071535347
Submit Date2025-05-13
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2025-06-09
Release Version1
George Poulogiannis George Poulogiannis
https://dx.doi.org/10.21228/M8X84J
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002458
Project DOI:doi: 10.21228/M8X84J
Project Title:Assessing the impact of SLC7A11 silencing in amoeboid disseminating cancer cells
Project Summary:Metabolomic analysis was performed to investigate changes in oxidative stress status in amoeboid cells. Using Liquid Chromatography-Mass Spectrometry (LC-MS), an amoeboid cancer cell line model and perturbations using siRNA or chemical compounds, we find that SLC7A11 protects cancer cells against oxidative stress.
Institute:The Institute of Cancer Research London
Department:Cell and Molecular Biology
Laboratory:Signalling and Cancer Metabolism
Last Name:Poulogiannis
First Name:George
Address:237 Fulham Road SW3 6JB LONDON
Email:George.poulogiannis@icr.ac.uk
Phone:+442071535347

Subject:

Subject ID:SU004061
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Strain Details:A375M2 (CVCL_C0RP)

Factors:

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

mb_sample_id local_sample_id Sample source Sample_type Treatment Labelled_GSH_Supplementation
SA445671DMSO 5A375M2 cancer cell line Cultured cells DMSO No
SA445672DMSO 4A375M2 cancer cell line Cultured cells DMSO No
SA445673DMSO 2A375M2 cancer cell line Cultured cells DMSO No
SA445674DMSO 1A375M2 cancer cell line Cultured cells DMSO No
SA445675BSO 1A375M2 cancer cell line Cultured cells L-BSO No
SA445676BSO 2A375M2 cancer cell line Cultured cells L-BSO No
SA445677BSO 3A375M2 cancer cell line Cultured cells L-BSO No
SA445678Menadione 2A375M2 cancer cell line Cultured cells Menadione No
SA445679Menadione 5A375M2 cancer cell line Cultured cells Menadione No
SA445680Menadione 4A375M2 cancer cell line Cultured cells Menadione No
SA445681Menadione 1A375M2 cancer cell line Cultured cells Menadione No
SA445695Scramble 5A375M2 cancer cell line Cultured cells siScramble No
SA445696Scramble 4A375M2 cancer cell line Cultured cells siScramble No
SA445697Scramble 3A375M2 cancer cell line Cultured cells siScramble No
SA445698Scramble 2A375M2 cancer cell line Cultured cells siScramble No
SA445699Scramble 1A375M2 cancer cell line Cultured cells siScramble No
SA445700Scramble + GSH 5A375M2 cancer cell line Cultured cells siScramble Yes
SA445701Scramble + GSH 3A375M2 cancer cell line Cultured cells siScramble Yes
SA445702Scramble + GSH 2A375M2 cancer cell line Cultured cells siScramble Yes
SA445703Scramble + GSH 1A375M2 cancer cell line Cultured cells siScramble Yes
SA445704Scramble + GSH 4A375M2 cancer cell line Cultured cells siScramble Yes
SA445685siSLC7A11 4A375M2 cancer cell line Cultured cells siSLC7A11 No
SA445686siSLC7A11 2A375M2 cancer cell line Cultured cells siSLC7A11 No
SA445687siSLC7A11 1A375M2 cancer cell line Cultured cells siSLC7A11 No
SA445688siSLC7A11 5A375M2 cancer cell line Cultured cells siSLC7A11 No
SA445689siSLC7A11 3A375M2 cancer cell line Cultured cells siSLC7A11 No
SA445690siSLC7A11 + GSH 4A375M2 cancer cell line Cultured cells siSLC7A11 Yes
SA445691siSLC7A11 + GSH 5A375M2 cancer cell line Cultured cells siSLC7A11 Yes
SA445692siSLC7A11 + GSH 1A375M2 cancer cell line Cultured cells siSLC7A11 Yes
SA445693siSLC7A11 + GSH 2A375M2 cancer cell line Cultured cells siSLC7A11 Yes
SA445694siSLC7A11 + GSH 3A375M2 cancer cell line Cultured cells siSLC7A11 Yes
SA445682Control 1A375M2 cancer cell line Cultured cells Water No
SA445683Control 2A375M2 cancer cell line Cultured cells Water No
SA445684Control 3A375M2 cancer cell line Cultured cells Water No
SA445661Scramble media 2A375M2 cancer cell line Culture media siScramble No
SA445662Scramble media 3A375M2 cancer cell line Culture media siScramble No
SA445663Scramble media 4A375M2 cancer cell line Culture media siScramble No
SA445664Scramble media 1A375M2 cancer cell line Culture media siScramble No
SA445665Scramble media 5A375M2 cancer cell line Culture media siScramble No
SA445666Scramble + GSH media 3A375M2 cancer cell line Culture media siScramble Yes
SA445667Scramble + GSH media 1A375M2 cancer cell line Culture media siScramble Yes
SA445668Scramble + GSH media 2A375M2 cancer cell line Culture media siScramble Yes
SA445669Scramble + GSH media 5A375M2 cancer cell line Culture media siScramble Yes
SA445670Scramble + GSH media 4A375M2 cancer cell line Culture media siScramble Yes
SA445651siSLC7A11 media 1A375M2 cancer cell line Culture media siSLC7A11 No
SA445652siSLC7A11 media 2A375M2 cancer cell line Culture media siSLC7A11 No
SA445653siSLC7A11 media 3A375M2 cancer cell line Culture media siSLC7A11 No
SA445654siSLC7A11 media 4A375M2 cancer cell line Culture media siSLC7A11 No
SA445655siSLC7A11 media 5A375M2 cancer cell line Culture media siSLC7A11 No
SA445656siSLC7A11 + GSH media 4A375M2 cancer cell line Culture media siSLC7A11 Yes
SA445657siSLC7A11 + GSH media 1A375M2 cancer cell line Culture media siSLC7A11 Yes
SA445658siSLC7A11 + GSH media 3A375M2 cancer cell line Culture media siSLC7A11 Yes
SA445659siSLC7A11 + GSH media 2A375M2 cancer cell line Culture media siSLC7A11 Yes
SA445660siSLC7A11 + GSH media 5A375M2 cancer cell line Culture media siSLC7A11 Yes
Showing results 1 to 54 of 54

Collection:

Collection ID:CO004054
Collection Summary:A375M2 (human melanoma cell line), A375M2 siScramble or A375M2 siSLC7A11 cells were counted and seeded at 250,000 cells per well in a 6-well plate and incubated for 48 hours to reach ~70-80% confluence. On the day of extraction, 6-well plates were placed on ice and 500μL media was collected in 1.5mL screw-cap vials and put on ice. Cells were then washed twice with cold 30mM N-Ethylmaleimide (NEM)-Phosphate Buffer Saline (PBS) (pH 7.4) and snap-frozen in liquid nitrogen then placed back on ice until extraction.
Collection Protocol Filename:GSH_GSSG_protocol.pdf
Sample Type:Cultured cells, Culture media
Storage Conditions:-80℃
Storage Vials:1.5ml screw cap-tubes
Collection Tube Temp:4

Treatment:

Treatment ID:TR004070
Treatment Summary:Cells were treated as follow for each of the experiment indicated in the metadata: 1) 250 µM Buthionine Sulfoximine in 1% FBS for 24h prior to extraction 2) 10µM menadione in 1% FBS for 24h prior to extraction 3) 10mM 13C2,15N -labeled GSH (Cambridge isotope, CNLM-6245) in 1% FBS media overnight prior to extraction.

Sample Preparation:

Sampleprep ID:SP004067
Sampleprep Summary:Cells and media were extracted using an extraction buffer composed of Methanol-Acetonitrile-Water (40:40:20) supplemented with 1mM NEM. For cells: 200uL of extraction was added to each well of a 6w plate. Cells were scraped, transferred into an Eppendorf tube. After spinning down for 10 min at 4*C, supernatant was transferred into an LC-MS V-shaped vial and stored at -20*C until analysis. For media: 200ul of media was mixed with 200ul of extraction buffer. After spinning down for 10 min at 4*C, supernatant was transferred into an LC-MS V-shaped vial and stored at -20*C until analysis.
Sampleprep Protocol Filename:GSH_GSSG_protocol.pdf

Combined analysis:

Analysis ID AN006447
Analysis type MS
Chromatography type Reversed phase
Chromatography system Agilent 1290 Infinity II
Column Agilent InfinityLab Poroshell 120 EC-C18 (150 x 3.0mm, 2.7um)
MS Type ESI
MS instrument type QTOF
MS instrument name Agilent 6546 QTOF
Ion Mode POSITIVE
Units Peak Area

Chromatography:

Chromatography ID:CH004893
Instrument Name:Agilent 1290 Infinity II
Column Name:Agilent InfinityLab Poroshell 120 EC-C18 (150 x 3.0mm, 2.7um)
Column Temperature:40
Flow Gradient:0min-1%B; 1.5min-1%B; 15min-80%B; 17min-99%B; 19min-99%B; 20min-1%B
Flow Rate:0.200mL/min
Solvent A:100% water; 0.1% Formic acid; 2.5uM Agilent InfinityLab Deactivator
Solvent B:100% acetonitrile; 0.1% Formic acid; 2.5uM Agilent InfinityLab Deactivator
Chromatography Type:Reversed phase

MS:

MS ID:MS006146
Analysis ID:AN006447
Instrument Name:Agilent 6546 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Agilent Masshunter Workstation Data Acquisition software was used for data acquisition. For data analysis, Agilent Profinder B.8.0.00 was used with a library of pre-determined retention times (+/- 0.20min shift) and accurate mass (+/- 15ppm shift) for metabolites of interests built using standards.
Ion Mode:POSITIVE
Capillary Voltage:3000
Dry Gas Flow:6l/min
Dry Gas Temp:275
Fragment Voltage:175
Nebulizer:20 psi
Octpole Voltage:750
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