#METABOLOMICS WORKBENCH gpoulogiannis_20250513_040453 DATATRACK_ID:5909 STUDY_ID:ST003925 ANALYSIS_ID:AN006447 PROJECT_ID:PR002458 VERSION 1 CREATED_ON May 20, 2025, 1:38 pm #PROJECT PR:PROJECT_TITLE Assessing the impact of SLC7A11 silencing in amoeboid disseminating cancer cells PR:PROJECT_SUMMARY Metabolomic analysis was performed to investigate changes in oxidative stress PR:PROJECT_SUMMARY status in amoeboid cells. Using Liquid Chromatography-Mass Spectrometry (LC-MS), PR:PROJECT_SUMMARY an amoeboid cancer cell line model and perturbations using siRNA or chemical PR:PROJECT_SUMMARY compounds, we find that SLC7A11 protects cancer cells against oxidative stress. PR:INSTITUTE The Institute of Cancer Research London PR:DEPARTMENT Cell and Molecular Biology PR:LABORATORY Signalling and Cancer Metabolism PR:LAST_NAME Poulogiannis PR:FIRST_NAME George PR:ADDRESS 237 Fulham Road SW3 6JB LONDON PR:EMAIL George.poulogiannis@icr.ac.uk PR:PHONE +442071535347 #STUDY ST:STUDY_TITLE Assessing the impact of SLC7A11 silencing on oxidative stress in amoeboid ST:STUDY_TITLE disseminating cancer cells ST:STUDY_SUMMARY Metabolomic analysis was performed to investigate changes in oxidative stress ST:STUDY_SUMMARY status in amoeboid cells. Oxidative stress limits metastasis and amoeboid cancer ST:STUDY_SUMMARY cells have been identified in a variety of cancers as a subset of metastatic ST:STUDY_SUMMARY cancer cells characterised by a high Rho-ROCK driven Myosin II activity. ST:STUDY_SUMMARY Amoeboid migrating cells require lower mitochondrial metabolism, while how they ST:STUDY_SUMMARY maintain low oxidative stress remains unclear. To interrogate the importance of ST:STUDY_SUMMARY SLC7A11 in oxidative stress regulation in amoeboid cells, two compounds ST:STUDY_SUMMARY (menadione and buthionine sulfoximine (BSO)) or an siRNA targeting SLC7A11 ST:STUDY_SUMMARY (siSLC7A11) were used. To assess the efficacy of these treatments, measurement ST:STUDY_SUMMARY of oxidised to reduced glutathione ratio (GSH/GSSG), oxidised to reduced ST:STUDY_SUMMARY nicotinamide adenine dinucleotide (NAD+/NADH) and cysteine levels using LC-MS ST:STUDY_SUMMARY was performed. Results indicate that treatment timings and concentration with ST:STUDY_SUMMARY menadione and BSO used throughout the study significantly reduce GSH/GSSG and ST:STUDY_SUMMARY NAD+/NADH ratio. Additionally, the effect of glutathione supplementation was ST:STUDY_SUMMARY tested in combination with siSLC7A11. Results indicate that SLC7A11 does not ST:STUDY_SUMMARY interfere with internalisation of glutathione supplemented in the media as the ST:STUDY_SUMMARY percentage of labelled glutathione was similar between siSLC7A11 and the siRNA ST:STUDY_SUMMARY control. ST:INSTITUTE The Institute of Cancer Research London ST:DEPARTMENT Cell and Molecular Biology ST:LABORATORY Signalling and Cancer Metabolism ST:LAST_NAME Poulogiannis ST:FIRST_NAME George ST:ADDRESS 237 Fulham Road SW3 6JB LONDON ST:EMAIL george.poulogiannis@icr.ac.uk ST:PHONE +442071535347 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS A375M2 (CVCL_C0RP) #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Control 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Water | Labelled_GSH_Supplementation:No Experiment_batch=1; RAW_FILE_NAME(Raw_File_Name)=Control 1.d SUBJECT_SAMPLE_FACTORS - Control 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Water | Labelled_GSH_Supplementation:No Experiment_batch=1; RAW_FILE_NAME(Raw_File_Name)=Control 2.d SUBJECT_SAMPLE_FACTORS - Control 3 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Water | Labelled_GSH_Supplementation:No Experiment_batch=1; RAW_FILE_NAME(Raw_File_Name)=Control 3.d SUBJECT_SAMPLE_FACTORS - BSO 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:L-BSO | Labelled_GSH_Supplementation:No Experiment_batch=1; RAW_FILE_NAME(Raw_File_Name)=BSO 1.d SUBJECT_SAMPLE_FACTORS - BSO 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:L-BSO | Labelled_GSH_Supplementation:No Experiment_batch=1; RAW_FILE_NAME(Raw_File_Name)=BSO 2.d SUBJECT_SAMPLE_FACTORS - BSO 3 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:L-BSO | Labelled_GSH_Supplementation:No Experiment_batch=1; RAW_FILE_NAME(Raw_File_Name)=BSO 3.d SUBJECT_SAMPLE_FACTORS - DMSO 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=DMSO 1.d SUBJECT_SAMPLE_FACTORS - DMSO 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=DMSO 2.d SUBJECT_SAMPLE_FACTORS - DMSO 4 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=DMSO 4.d SUBJECT_SAMPLE_FACTORS - DMSO 5 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=DMSO 5.d SUBJECT_SAMPLE_FACTORS - Menadione 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=Menadione 1.d SUBJECT_SAMPLE_FACTORS - Menadione 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=Menadione 2.d SUBJECT_SAMPLE_FACTORS - Menadione 4 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=Menadione 4.d SUBJECT_SAMPLE_FACTORS - Menadione 5 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Experiment_batch=2; RAW_FILE_NAME(Raw_File_Name)=Menadione 5.d SUBJECT_SAMPLE_FACTORS - Scramble 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble 1.d SUBJECT_SAMPLE_FACTORS - Scramble 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble 2.d SUBJECT_SAMPLE_FACTORS - Scramble 3 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble 3.d SUBJECT_SAMPLE_FACTORS - Scramble 4 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble 4.d SUBJECT_SAMPLE_FACTORS - Scramble 5 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble 5.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH 1.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH 2.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH 3 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH 3.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH 4 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH 4.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH 5 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH 5.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 1.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 2.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 3 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 3.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 4 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 4.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 5 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 5.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH 1 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH 1.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH 2 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH 2.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH 3 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH 3.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH 4 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH 4.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH 5 Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH 5.d SUBJECT_SAMPLE_FACTORS - Scramble media 1 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble media 1.d SUBJECT_SAMPLE_FACTORS - Scramble media 2 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble media 2.d SUBJECT_SAMPLE_FACTORS - Scramble media 3 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble media 3.d SUBJECT_SAMPLE_FACTORS - Scramble media 4 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble media 4.d SUBJECT_SAMPLE_FACTORS - Scramble media 5 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble media 5.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH media 1 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH media 1.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH media 2 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH media 2.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH media 3 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH media 3.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH media 4 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH media 4.d SUBJECT_SAMPLE_FACTORS - Scramble + GSH media 5 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=Scramble + GSH media 5.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 media 1 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 media 1.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 media 2 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 media 2.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 media 3 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 media 3.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 media 4 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 media 4.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 media 5 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 media 5.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH media 1 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH media 1.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH media 2 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH media 2.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH media 3 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH media 3.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH media 4 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH media 4.d SUBJECT_SAMPLE_FACTORS - siSLC7A11 + GSH media 5 Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Experiment_batch=3; RAW_FILE_NAME(Raw_File_Name)=siSLC7A11 + GSH media 5.d #COLLECTION CO:COLLECTION_SUMMARY A375M2 (human melanoma cell line), A375M2 siScramble or A375M2 siSLC7A11 cells CO:COLLECTION_SUMMARY were counted and seeded at 250,000 cells per well in a 6-well plate and CO:COLLECTION_SUMMARY incubated for 48 hours to reach ~70-80% confluence. On the day of extraction, CO:COLLECTION_SUMMARY 6-well plates were placed on ice and 500μL media was collected in 1.5mL CO:COLLECTION_SUMMARY screw-cap vials and put on ice. Cells were then washed twice with cold 30mM CO:COLLECTION_SUMMARY N-Ethylmaleimide (NEM)-Phosphate Buffer Saline (PBS) (pH 7.4) and snap-frozen in CO:COLLECTION_SUMMARY liquid nitrogen then placed back on ice until extraction. CO:COLLECTION_PROTOCOL_FILENAME GSH_GSSG_protocol.pdf CO:SAMPLE_TYPE Cultured cells, Culture media CO:STORAGE_CONDITIONS -80℃ CO:STORAGE_VIALS 1.5ml screw cap-tubes CO:COLLECTION_TUBE_TEMP 4 #TREATMENT TR:TREATMENT_SUMMARY Cells were treated as follow for each of the experiment indicated in the TR:TREATMENT_SUMMARY metadata: 1) 250 µM Buthionine Sulfoximine in 1% FBS for 24h prior to TR:TREATMENT_SUMMARY extraction 2) 10µM menadione in 1% FBS for 24h prior to extraction 3) 10mM TR:TREATMENT_SUMMARY 13C2,15N -labeled GSH (Cambridge isotope, CNLM-6245) in 1% FBS media overnight TR:TREATMENT_SUMMARY prior to extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells and media were extracted using an extraction buffer composed of SP:SAMPLEPREP_SUMMARY Methanol-Acetonitrile-Water (40:40:20) supplemented with 1mM NEM. For cells: SP:SAMPLEPREP_SUMMARY 200uL of extraction was added to each well of a 6w plate. Cells were scraped, SP:SAMPLEPREP_SUMMARY transferred into an Eppendorf tube. After spinning down for 10 min at 4*C, SP:SAMPLEPREP_SUMMARY supernatant was transferred into an LC-MS V-shaped vial and stored at -20*C SP:SAMPLEPREP_SUMMARY until analysis. For media: 200ul of media was mixed with 200ul of extraction SP:SAMPLEPREP_SUMMARY buffer. After spinning down for 10 min at 4*C, supernatant was transferred into SP:SAMPLEPREP_SUMMARY an LC-MS V-shaped vial and stored at -20*C until analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME GSH_GSSG_protocol.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C18 (150 x 3.0mm, 2.7um) CH:SOLVENT_A 100% water; 0.1% Formic acid; 2.5uM Agilent InfinityLab Deactivator CH:SOLVENT_B 100% acetonitrile; 0.1% Formic acid; 2.5uM Agilent InfinityLab Deactivator CH:FLOW_GRADIENT 0min-1%B; 1.5min-1%B; 15min-80%B; 17min-99%B; 19min-99%B; 20min-1%B CH:FLOW_RATE 0.200mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Signalling and Cancer Metabolism AN:ACQUISITION_DATE 02/2025 AN:DATA_FORMAT .d #MS MS:INSTRUMENT_NAME Agilent 6546 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Agilent Masshunter Workstation Data Acquisition software was used for data MS:MS_COMMENTS acquisition. For data analysis, Agilent Profinder B.8.0.00 was used with a MS:MS_COMMENTS library of pre-determined retention times (+/- 0.20min shift) and accurate mass MS:MS_COMMENTS (+/- 15ppm shift) for metabolites of interests built using standards. MS:CAPILLARY_VOLTAGE 3000 MS:DRY_GAS_FLOW 6l/min MS:DRY_GAS_TEMP 275 MS:FRAGMENT_VOLTAGE 175 MS:NEBULIZER 20 psi MS:OCTPOLE_VOLTAGE 750 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples Control 1 Control 2 Control 3 BSO 1 BSO 2 BSO 3 DMSO 1 DMSO 2 DMSO 4 DMSO 5 Menadione 1 Menadione 2 Menadione 4 Menadione 5 Scramble + GSH 1 Scramble + GSH 2 Scramble + GSH 3 Scramble + GSH 4 Scramble + GSH 5 Scramble 1 Scramble 2 Scramble 3 Scramble 4 Scramble 5 siSLC7A11 + GSH 1 siSLC7A11 + GSH 2 siSLC7A11 + GSH 3 siSLC7A11 + GSH 4 siSLC7A11 + GSH 5 siSLC7A11 1 siSLC7A11 2 siSLC7A11 3 siSLC7A11 4 siSLC7A11 5 Scramble media 1 Scramble media 2 Scramble media 3 Scramble media 4 Scramble media 5 siSLC7A11 media 1 siSLC7A11 media 2 siSLC7A11 media 3 siSLC7A11 media 4 siSLC7A11 media 5 Scramble + GSH media 1 Scramble + GSH media 2 Scramble + GSH media 3 Scramble + GSH media 4 Scramble + GSH media 5 siSLC7A11 + GSH media 1 siSLC7A11 + GSH media 2 siSLC7A11 + GSH media 3 siSLC7A11 + GSH media 4 siSLC7A11 + GSH media 5 Factors Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Water | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Water | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Water | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:L-BSO | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:L-BSO | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:L-BSO | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:DMSO | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:Menadione | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Cultured cells | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:No Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siScramble | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes Sample source:A375M2 cancer cell line | Sample_type:Culture media | Treatment:siSLC7A11 | Labelled_GSH_Supplementation:Yes NADH 73355.99536 52093.92175 59150.04248 47207.81372 57404.94592 55605.97009 30184.68347 40321.59821 38350.45593 33598.21063 47313.72284 56687.65088 59094.82666 47637.82623 NAD+ 240692.3008 191264.6025 188176.8589 77079.29321 106734.1584 103414.1445 80040.03003 72958.74536 70327.69043 81294.59204 155102.9385 65176.33789 63140.7821 62918.39923 GS-NEM 85603627.88 82309039 91147906 6981822.5 10071240.44 9359025.375 194618924 192986776.5 197718591.5 195306473.3 200428728.3 209847233.8 214113860 203447804 185737144 566922.574 183091676 580041.727 172909482 194991042 225814288 222105815 220150560 219496014 175150904 182830952 188369538 187306592 44780468.8 213321029 219576876 226745935 219931628 220701983 3467034.156 3571073.098 3355895.281 3257839.044 3193233.102 549263.5098 462534.25 723740.958 451657.0391 688945.5181 1984048.258 2031823.234 1879244.203 1922766.012 1900780.988 1812177.406 1859440.828 1803744.316 1867829.188 1958484.992 GSSG 70923.26465 103426.0195 81802.73486 75143.16162 109687.073 96854.03369 43582.91156 40496.01343 39061.35645 37428.02148 85792.14941 71302.39221 85454.43787 76561.2915 Cysteine-NEM 2247978.03 33386.5077 2564659.93 26766.7466 2648916.02 1790998.3 2130057.33 1987156.92 2084066.1 1970294.28 2231023.81 2198331.78 2409602.5 2138754.31 2243807.59 1050930.25 992177.305 1132676.26 1311679.84 911204.32 5254159.5 5309994.734 2858943.328 5053443.688 5506854.953 1746188.504 1497645.152 1896599.914 1639976.387 1960879.871 10019324.91 10356300 10434043.09 10329967.31 9970988.063 10925073.59 10825367.5 10597939.66 9361187.719 10040308.2 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Formula Mass RT PubChemID KEGG ID CAS NADH C21 H29 N7 O14 P2 665.1248 6.89 439153 C00004 58-68-4 NAD+ C21 H28 N7 O14 P2 664.1169 6.89 5893 C00003 53-84-9 GS-NEM C16 H24 N4 O8 S 432.1314 8.21 443150 C11370 97908-43-5 GSSG C20 H32 N6 O12 S2 612.152 6.91 65359 C00127 27025-41-8 Cysteine-NEM C9 H14 N2 O4 S 246.0674 7.3 6453933 NA 60218-98-6 METABOLITES_END #END