#METABOLOMICS WORKBENCH wanglgene_20241206_115101 DATATRACK_ID:5438 STUDY_ID:ST003625 ANALYSIS_ID:AN005955 PROJECT_ID:PR002208 VERSION 1 CREATED_ON December 12, 2024, 6:33 pm #PROJECT PR:PROJECT_TITLE NRF2 supports non-small cell lung cancer growth independently of PR:PROJECT_TITLE CBP/p300-enhanced glutathione synthesis PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress responsive PR:PROJECT_SUMMARY transcription factor that is mutationally activated in a subset (~25%) of PR:PROJECT_SUMMARY clinically-aggressive non-small cell lung cancers (NSCLC). Mechanistic insight PR:PROJECT_SUMMARY into drivers of the NRF2 dependency remains poorly understood. Here, we defined PR:PROJECT_SUMMARY a novel NRF2 target gene set linked to NRF2-dependency in cancer cell lines, and PR:PROJECT_SUMMARY observed that a significant portion of these genes is devoid of PR:PROJECT_SUMMARY promoter-proximal NRF2. Using integrated genomic analyses, we characterized PR:PROJECT_SUMMARY extensive NRF2-dependent enhancer RNA (eRNA) synthesis and NRF2-mediated H3K27ac PR:PROJECT_SUMMARY deposition at proximal and distal enhancer regions regulating these genes. While PR:PROJECT_SUMMARY CBP/p300 is a well-validated direct interaction partner of NRF2 with prominent PR:PROJECT_SUMMARY functions at enhancers, we report that this interaction is not required for PR:PROJECT_SUMMARY NRF2-dependent NSCLC cell growth, indicating that NRF2 can sustain sufficient PR:PROJECT_SUMMARY transcriptional activity in the absence of CBP/p300 coactivation. Broad PR:PROJECT_SUMMARY metabolic profiling established a primary role for CBP/p300 in NRF2-dependent PR:PROJECT_SUMMARY accumulation of glutathione and glutathione-related metabolites. While redox PR:PROJECT_SUMMARY homeostasis via enhanced glutathione production is commonly associated with the PR:PROJECT_SUMMARY normal physiological role of NRF2, collectively our results suggest that PR:PROJECT_SUMMARY NRF2-dependent cancer cell growth does not require this enhanced glutathione PR:PROJECT_SUMMARY production. PR:INSTITUTE Genentech Inc. PR:LAST_NAME Wang PR:FIRST_NAME Mike Lingjue PR:ADDRESS 1 DNA Way, South San Francisco, CA 94404 PR:EMAIL wang.mike@gene.com PR:PHONE 650-245-7991 PR:CONTRIBUTORS Ryan J. Conrad, James A. Mondo, Mike Lingjue Wang, Peter S. Liu, Zijuan Lai, PR:CONTRIBUTORS Feroza K Choudhury, Qingling Li, Weng Ruh Wong, James Lee, Frances Shanahan, Eva PR:CONTRIBUTORS Lin, Scott Martin, Joachim Rudolph, John G. Moffat, Dewakar Sangaraju, Wendy PR:CONTRIBUTORS Sandoval, Timothy Sterne-Weiler, Scott A. Foster #STUDY ST:STUDY_TITLE NRF2 supports non-small cell lung cancer growth independently of ST:STUDY_TITLE CBP/p300-enhanced glutathione synthesis: Using 1,2-13C-glucose to measure PPP ST:STUDY_TITLE Flux in A549 cells (Part 3 of 3) ST:STUDY_SUMMARY The study aims at validating the hypothesis that NRF2 mediates the redox ST:STUDY_SUMMARY homeostasis through regulating oxidative pentose phosphate pathway. The study ST:STUDY_SUMMARY uses 1,2-13C-glucose to label A549 cells with or without NRF2 and the oxidative ST:STUDY_SUMMARY PPP can be measured by looking at ratios of [M+1] and [M+1]+[M+2] lactates. ST:STUDY_SUMMARY Briefly, metabolites were extracted from cells and media with cold ST:STUDY_SUMMARY ACN/MeOH/water mixture and analyzed by LC-HRMS to determine glucose uptake rate ST:STUDY_SUMMARY and lactate isotopologues. The result shows that PPP flux is reduced in the ST:STUDY_SUMMARY absence of NRF2. ST:INSTITUTE Genentech Inc. ST:LAST_NAME Wang ST:FIRST_NAME Mike ST:ADDRESS 1 DNA Way, South San Francisco, CA 94080, USA ST:EMAIL wang.mike@gene.com ST:PHONE 6502457991 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS A549 SU:SPECIES_GROUP Mammals #FACTORS #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 - CellA1 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=0.416; RAW_FILE_NAME(Raw data)=20240809_025_Amide-m2_negddMS2_A1_cell SUBJECT_SAMPLE_FACTORS - CellA2 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=0.416; RAW_FILE_NAME(Raw data)=20240809_018_Amide-m2_negddMS2_A2_cell SUBJECT_SAMPLE_FACTORS - CellA3 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=0.416; RAW_FILE_NAME(Raw data)=20240809_008_Amide-m2_negddMS2_A3_cell SUBJECT_SAMPLE_FACTORS - CellA4 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=0.416; RAW_FILE_NAME(Raw data)=20240809_022_Amide-m2_negddMS2_A4_cell SUBJECT_SAMPLE_FACTORS - CellA5 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=0.416; RAW_FILE_NAME(Raw data)=20240809_013_Amide-m2_negddMS2_A5_cell SUBJECT_SAMPLE_FACTORS - CellB1 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=0.414; RAW_FILE_NAME(Raw data)=20240809_021_Amide-m2_negddMS2_B1_cell SUBJECT_SAMPLE_FACTORS - CellB2 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=0.414; RAW_FILE_NAME(Raw data)=20240809_017_Amide-m2_negddMS2_B2_cell SUBJECT_SAMPLE_FACTORS - CellB3 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=0.414; RAW_FILE_NAME(Raw data)=20240809_014_Amide-m2_negddMS2_B3_cell SUBJECT_SAMPLE_FACTORS - CellB4 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=0.414; RAW_FILE_NAME(Raw data)=20240809_007_Amide-m2_negddMS2_B4_cell SUBJECT_SAMPLE_FACTORS - CellB5 Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=0.414; RAW_FILE_NAME(Raw data)=20240809_011_Amide-m2_negddMS2_B5_cell SUBJECT_SAMPLE_FACTORS - CellC1 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Cell Count (1E6)=0.467; RAW_FILE_NAME(Raw data)=20240809_020_Amide-m2_negddMS2_C1_cell SUBJECT_SAMPLE_FACTORS - CellC2 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Cell Count (1E6)=0.467; RAW_FILE_NAME(Raw data)=20240809_024_Amide-m2_negddMS2_C2_cell SUBJECT_SAMPLE_FACTORS - CellC3 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Cell Count (1E6)=0.467; RAW_FILE_NAME(Raw data)=20240809_010_Amide-m2_negddMS2_C3_cell SUBJECT_SAMPLE_FACTORS - CellC4 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Cell Count (1E6)=0.467; RAW_FILE_NAME(Raw data)=20240809_009_Amide-m2_negddMS2_C4_cell SUBJECT_SAMPLE_FACTORS - CellC5 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Cell Count (1E6)=0.467; RAW_FILE_NAME(Raw data)=20240809_019_Amide-m2_negddMS2_C5_cell SUBJECT_SAMPLE_FACTORS - CellD1 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=0.455; RAW_FILE_NAME(Raw data)=20240809_026_Amide-m2_negddMS2_D1_cell SUBJECT_SAMPLE_FACTORS - CellD2 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=0.455; RAW_FILE_NAME(Raw data)=20240809_006_Amide-m2_negddMS2_D2_cell SUBJECT_SAMPLE_FACTORS - CellD3 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=0.455; RAW_FILE_NAME(Raw data)=20240809_023_Amide-m2_negddMS2_D3_cell SUBJECT_SAMPLE_FACTORS - CellD4 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=0.455; RAW_FILE_NAME(Raw data)=20240809_005_Amide-m2_negddMS2_D4_cell SUBJECT_SAMPLE_FACTORS - CellD5 Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=0.455; RAW_FILE_NAME(Raw data)=20240809_012_Amide-m2_negddMS2_D5_cell SUBJECT_SAMPLE_FACTORS - MediaA1pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_082_Amide-m3_negddMS2_A1_pre SUBJECT_SAMPLE_FACTORS - MediaA2pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_047_Amide-m3_negddMS2_A2_pre SUBJECT_SAMPLE_FACTORS - MediaA3pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_049_Amide-m3_negddMS2_A3_pre SUBJECT_SAMPLE_FACTORS - MediaA4pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_078_Amide-m3_negddMS2_A4_pre SUBJECT_SAMPLE_FACTORS - MediaA5pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_070_Amide-m3_negddMS2_A5_pre SUBJECT_SAMPLE_FACTORS - MediaB1pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_061_Amide-m3_negddMS2_B1_pre SUBJECT_SAMPLE_FACTORS - MediaB2pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_087_Amide-m3_negddMS2_B2_pre SUBJECT_SAMPLE_FACTORS - MediaB3pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_076_Amide-m3_negddMS2_B3_pre SUBJECT_SAMPLE_FACTORS - MediaB4pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_073_Amide-m3_negddMS2_B4_pre SUBJECT_SAMPLE_FACTORS - MediaB5pre Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_050_Amide-m3_negddMS2_B5_pre SUBJECT_SAMPLE_FACTORS - MediaC1pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_088_Amide-m3_negddMS2_C1_pre SUBJECT_SAMPLE_FACTORS - MediaC2pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_045_Amide-m3_negddMS2_C2_pre SUBJECT_SAMPLE_FACTORS - MediaC3pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_058_Amide-m3_negddMS2_C3_pre SUBJECT_SAMPLE_FACTORS - MediaC4pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_079_Amide-m3_negddMS2_C4_pre SUBJECT_SAMPLE_FACTORS - MediaC5pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_080_Amide-m3_negddMS2_C5_pre SUBJECT_SAMPLE_FACTORS - MediaD1pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_062_Amide-m3_negddMS2_D1_pre SUBJECT_SAMPLE_FACTORS - MediaD2pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_069_Amide-m3_negddMS2_D2_pre SUBJECT_SAMPLE_FACTORS - MediaD3pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_064_Amide-m3_negddMS2_D3_pre SUBJECT_SAMPLE_FACTORS - MediaD4pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_074_Amide-m3_negddMS2_D4_pre SUBJECT_SAMPLE_FACTORS - MediaD5pre Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_085_Amide-m3_negddMS2_D5_pre SUBJECT_SAMPLE_FACTORS - MediaA1post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_052_Amide-m3_negddMS2_A1_after SUBJECT_SAMPLE_FACTORS - MediaA2post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_075_Amide-m3_negddMS2_A2_after SUBJECT_SAMPLE_FACTORS - MediaA3post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_086_Amide-m3_negddMS2_A3_after SUBJECT_SAMPLE_FACTORS - MediaA4post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_046_Amide-m3_negddMS2_A4_after SUBJECT_SAMPLE_FACTORS - MediaA5post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_048_Amide-m3_negddMS2_A5_after SUBJECT_SAMPLE_FACTORS - MediaB1post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_055_Amide-m3_negddMS2_B1_after SUBJECT_SAMPLE_FACTORS - MediaB2post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_083_Amide-m3_negddMS2_B2_after SUBJECT_SAMPLE_FACTORS - MediaB3post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_063_Amide-m3_negddMS2_B3_after SUBJECT_SAMPLE_FACTORS - MediaB4post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_072_Amide-m3_negddMS2_B4_after SUBJECT_SAMPLE_FACTORS - MediaB5post Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_084_Amide-m3_negddMS2_B5_after SUBJECT_SAMPLE_FACTORS - MediaC1post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_057_Amide-m3_negddMS2_C1_after SUBJECT_SAMPLE_FACTORS - MediaC2post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_051_Amide-m3_negddMS2_C2_after SUBJECT_SAMPLE_FACTORS - MediaC3post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_077_Amide-m3_negddMS2_C3_after SUBJECT_SAMPLE_FACTORS - MediaC4post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_054_Amide-m3_negddMS2_C4_after SUBJECT_SAMPLE_FACTORS - MediaC5post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_056_Amide-m3_negddMS2_C5_after SUBJECT_SAMPLE_FACTORS - MediaD1post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_059_Amide-m3_negddMS2_D1_after SUBJECT_SAMPLE_FACTORS - MediaD2post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_081_Amide-m3_negddMS2_D2_after SUBJECT_SAMPLE_FACTORS - MediaD3post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_053_Amide-m3_negddMS2_D3_after SUBJECT_SAMPLE_FACTORS - MediaD4post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_060_Amide-m3_negddMS2_D4_after SUBJECT_SAMPLE_FACTORS - MediaD5post Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Cell Count (1E6)=na; RAW_FILE_NAME(Raw data)=20240814_071_Amide-m3_negddMS2_D5_after #COLLECTION CO:COLLECTION_SUMMARY All cell lines were obtained from the Genentech cell bank (Yu et al, 2015) and CO:COLLECTION_SUMMARY grown in RPMI or DMEM supplemented with 10% FBS, 1% glutamine, and 1% CO:COLLECTION_SUMMARY penicillin-streptomycin at 37°C, 5% CO2. Cell lines were confirmed via STR CO:COLLECTION_SUMMARY profiling and were mycoplasma free. A549 and A549shNRF2-BIND rescue cell lines CO:COLLECTION_SUMMARY were generated using the Piggybac system. Donor constructs were co-transfected CO:COLLECTION_SUMMARY with transposase (Ding et al, 2005) at a 4:1 ratio using Lipofetamine 3000 CO:COLLECTION_SUMMARY (Thermo) according to manufacturer’s protocol. 3-days post-transfection, CO:COLLECTION_SUMMARY puromycin selection (1µg/mL) was initiated and was performed for ~2 weeks prior CO:COLLECTION_SUMMARY to experimentation. A549shNRF2-BIND rescue cells represent polyclonal pools. CO:COLLECTION_SUMMARY Cell viability was measured using CellTiter-Glo® Luminescent Cell Viability CO:COLLECTION_SUMMARY Assay (Promega) using the Envision 2103 Plate Reader (Promega). Cell growth CO:COLLECTION_SUMMARY curves were generated using the IncuCyte System (Sartorius) according to the CO:COLLECTION_SUMMARY manufacturer’s protocol. Curve fits were performed in Prism. Clonogenic assays CO:COLLECTION_SUMMARY were performed by washing cells in PBS, staining with 0.5% crystal violet CO:COLLECTION_SUMMARY solution (Sigma HT90132) for 5 min, and washing 3x with PBS. Four replicates of CO:COLLECTION_SUMMARY 20-30 million A549shNRF2-BIND-luc, WT NRF2 or Neh4/5mut cells were treated with CO:COLLECTION_SUMMARY 2.5ng/mL dox for 48h. For labeling, cells were grown in RPMI media with CO:COLLECTION_SUMMARY unlabeled glucose and 10 % FBS for 36 hours and replenished with RPMI media CO:COLLECTION_SUMMARY containing 11.11 mM [1,2-13C]-glucose and incubated for 14.5 hours. Aliquots of CO:COLLECTION_SUMMARY media were taken at the beginning and end of labeling to measure glucose CO:COLLECTION_SUMMARY consumption rate. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY No treatment was administered and this is a study based on genetic variants. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, cells were harvested by washing with PBS twice and quenched on dry-ice SP:SAMPLEPREP_SUMMARY by adding acidified 2:2:1 ACN/MeOH/water with spiked-in 15N5-ADP (Sigma, St. SP:SAMPLEPREP_SUMMARY Louis, MO, USA) as an internal standard. 2M ammonium bicarbonate was then added SP:SAMPLEPREP_SUMMARY to adjust the pH to 8 (22:3 v/v). The extract was freeze-thawed by liquid SP:SAMPLEPREP_SUMMARY nitrogen and sonicated three times and centrifuged at 15,000 rpm for 15 minutes. SP:SAMPLEPREP_SUMMARY The supernatant was transferred and subjected to LC-MS analysis. The extract was SP:SAMPLEPREP_SUMMARY kept on ice in all steps. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Shimadzu Nexera HPLC CH:COLUMN_NAME Waters XBridge BEH Amide (100 x 2.1mm,2.5um) CH:SOLVENT_A 5% acetonitrile/95% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide CH:SOLVENT_B 95% acetonitrile/5% water CH:FLOW_GRADIENT 0-1 min 90% B, 4 min 50% B, 10-11.5 min 20% B, 12.5-17 min 90% B CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Samples were run in negative mode. The Q Exactive Plus Mass Spectrometer was MS:MS_COMMENTS operated with the following parameters: Sheath gas flow rate, 50 units; Aux gas MS:MS_COMMENTS flow rate, 13 units; Aux gas temperature, 425 °C; Capillary temperature, MS:MS_COMMENTS 263°C; Spray voltage, -2500V; Scan mode, MS2 scan with data-dependent MS/MS MS:MS_COMMENTS acquisition. In Full MS scan, scan range is 60-900 m/z; resolution is 70,000; MS:MS_COMMENTS AGC target, 1×e^6; Maximum IT, 100 ms. In ddMS2 scan, top 5 ions are selected MS:MS_COMMENTS with an isolation window of 1.0 m/z; resolution is 17,500; AGC target, 5×e^4; MS:MS_COMMENTS Maximum IT, 50 ms. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion Count MS_METABOLITE_DATA_START Samples CellA1 CellA2 CellA3 CellA4 CellA5 CellB1 CellB2 CellB3 CellB4 CellB5 CellC1 CellC2 CellC3 CellC4 CellC5 CellD1 CellD2 CellD3 CellD4 CellD5 MediaA1pre MediaA2pre MediaA3pre MediaA4pre MediaA5pre MediaB1pre MediaB2pre MediaB3pre MediaB4pre MediaB5pre MediaC1pre MediaC2pre MediaC3pre MediaC4pre MediaC5pre MediaD1pre MediaD2pre MediaD3pre MediaD4pre MediaD5pre MediaA1post MediaA2post MediaA3post MediaA4post MediaA5post MediaB1post MediaB2post MediaB3post MediaB4post MediaB5post MediaC1post MediaC2post MediaC3post MediaC4post MediaC5post MediaD1post MediaD2post MediaD3post MediaD4post MediaD5post Factors Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Human Lung Cancer Cell | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:DMSO Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Human Lung Cancer Cell | CellType:WT | Treatment:KEAP1 inhibitor Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox- Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:A549 Cell Culture Media | CellType:shNRF2-Luciferase | Treatment:Dox+ Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:DMSO Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Sample source:H441 Cell Culture Media | CellType:WT | Treatment:KEAP1 inhibitor Lactic acid_M0 2750786560 2699554304 1610880768 2925284608 2199422720 1252918912 1301804160 1034859968 1027041472 1148970112 2495118336 2978193664 1992508672 2100062848 3300756480 3429706496 2347056640 4426449408 2271304960 2821887488 na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na Lactic acid_M1 145601856 140041920 80982128 155462624 113626184 62838204 64904732 49979256 50020696 55141340 131347600 157721408 99643728 105134528 169780288 208656400 132154504 259239696 127073976 156458544 na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na Lactic acid_M2 1553611136 1556347904 843943296 1718810368 1186054400 708983680 734573568 549833920 529817344 621138304 1348240384 1658899456 984118976 1021014720 1754745984 1964972288 1181471488 2414096640 1096395392 1352649728 na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na Lactic acid_M3 21465388 29188252 35813760 22331302 44681164 11199226 19551164 21690928 23431708 25023750 36206484 34316180 58054748 65462300 55974440 45042364 80480256 53138572 81044528 94363720 na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na Glucose na na na na na na na na na na na na na na na na na na na na 54656194 50067752 48890713 48112916 50767160 46482816 58628882 40566261 47360795 32021939 44429 58870699 56189929 58175164 52766702 45925040 53670102 51559055 48298340 47652183 35413255 34223023 37536526 30916240 36453139 44382650 46602446 42070893 46278403 46235542 37873683 32565538 24778839 40799966 35317964 38801938 34630271 34846134 35531248 35780096 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Ion Adduct Formula Exact mass Super class Main class Sub class Lactic acid_M0 [M-H]- C3H6O3 90.0317 Organic acids Short-chain acids Short-chain acids Lactic acid_M1 [M-H]- C3H6O3 91.03505 Organic acids Short-chain acids Short-chain acids Lactic acid_M2 [M-H]- C3H6O3 92.0384 Organic acids Short-chain acids Short-chain acids Lactic acid_M3 [M-H]- C3H6O3 93.04175 Organic acids Short-chain acids Short-chain acids Glucose [M-H]- C6H12O6 180.0634 Carbohydrates Monosaccharides Hexoses METABOLITES_END #END