{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003398","ANALYSIS_ID":"AN005578","PROJECT_ID":"PR002104","VERSION":"1","CREATED_ON":"August 12, 2024, 5:29 pm"},

"PROJECT":{"PROJECT_TITLE":"Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis","PROJECT_TYPE":"Biology","PROJECT_SUMMARY":"The integrated stress response (ISR) enables cells to cope with a variety of insults, but its specific contribution to downstream cellular outputs remains unclear. Using a synthetic tool, we selectively activate the ISR without co-activation of parallel pathways and define the resulting cellular state with multi-omics profiling. We identify time- and dose-dependent gene expression modules, with ATF4 driving only a small but sensitive subgroup that includes amino acid metabolic enzymes. This ATF4 response affects cellular bioenergetics, rerouting carbon utilization towards amino acid production and away from the tricarboxylic acid cycle and fatty acid synthesis. We also find an ATF4-independent reorganization of the lipidome that promotes DGAT-dependent triglyceride synthesis and accumulation of lipid droplets. While DGAT1 is the main driver of lipid droplet biogenesis, DGAT2 plays an essential role in buffering stress and maintaining cell survival. Together, we demonstrate the sufficiency of the ISR in promoting a previously unappreciated metabolic state.","INSTITUTE":"Calico Life Sciences","DEPARTMENT":"Department of Mass Spectrometry-Technology Lab","LABORATORY":"Metabolomics Lab","LAST_NAME":"Vu","FIRST_NAME":"Ngoc","ADDRESS":"1130 Veterans BLVD, South San Francisco, CA 94080","EMAIL":"ngochmvu@gmail.com","PHONE":"6504205430","PUBLICATIONS":"Labbe, Lebon & King et al., Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis.Nat Comm.2024"},

"STUDY":{"STUDY_TITLE":"Specific activation of the integrated stress response (ISR) uncovers regulation of lipid droplet biogenesis","STUDY_TYPE":"Biology","STUDY_SUMMARY":"U2OS cells were treated with Dimerizer-PERK for 0h,1h,2h,4h,8h and 24h. Lipidomics analysis using LC-MS was performed on these samples to understand the regulation of cellular lipidome upon ISR activation","INSTITUTE":"Calico Life Sciences","DEPARTMENT":"Department of Mass Spectrometry-Technology Lab","LABORATORY":"Metabolomics Lab","LAST_NAME":"Vu","FIRST_NAME":"Ngoc","ADDRESS":"1130 Veterans BLVD, South San Francisco, CA 94080","EMAIL":"ngoc@calicolabs.com","PHONE":"650-420-5430","NUM_GROUPS":"6","TOTAL_SUBJECTS":"18"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_STRAIN_DETAILS":"U2OS","CELL_COUNTS":"5e5"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"0h_1",
"Factors":{"Time":"0","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"1_WT_Dim_0h_1.mzML","condition #":"1","replicate #":"1","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"0h_2",
"Factors":{"Time":"0","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"2_WT_Dim_0h_2.mzML","condition #":"1","replicate #":"2","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"0h_3",
"Factors":{"Time":"0","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"3_WT_Dim_0h_3.mzML","condition #":"1","replicate #":"3","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"1h_1",
"Factors":{"Time":"1","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"4_WT_Dim_1h_1.mzML","condition #":"2","replicate #":"1","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"1h_2",
"Factors":{"Time":"1","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"5_WT_Dim_1h_2.mzML","condition #":"2","replicate #":"2","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"1h_3",
"Factors":{"Time":"1","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"6_WT_Dim_1h_3.mzML","condition #":"2","replicate #":"3","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"2h_1",
"Factors":{"Time":"2","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"7_WT_Dim_2h_1.mzML","condition #":"3","replicate #":"1","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"2h_2",
"Factors":{"Time":"2","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"8_WT_Dim_2h_2.mzML","condition #":"3","replicate #":"2","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"2h_3",
"Factors":{"Time":"2","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"9_WT_Dim_2h_3.mzML","condition #":"3","replicate #":"3","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"4h_1",
"Factors":{"Time":"4","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"10_WT_Dim_4h_1.mzML","condition #":"4","replicate #":"1","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"4h_2",
"Factors":{"Time":"4","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"11_WT_Dim_4h_2.mzML","condition #":"4","replicate #":"2","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"4h_3",
"Factors":{"Time":"4","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"12_WT_Dim_4h_3.mzML","condition #":"4","replicate #":"3","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"8h_1",
"Factors":{"Time":"8","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"13_WT_Dim_8h_1.mzML","condition #":"5","replicate #":"1","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"8h_2",
"Factors":{"Time":"8","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"14_WT_Dim_8h_2.mzML","condition #":"5","replicate #":"2","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"8h_3",
"Factors":{"Time":"8","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"15_WT_Dim_8h_3.mzML","condition #":"5","replicate #":"3","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"24h_1",
"Factors":{"Time":"24","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"16_WT_Dim_24h_1.mzML","condition #":"6","replicate #":"1","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"24h_2",
"Factors":{"Time":"24","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"17_WT_Dim_24h_2.mzML","condition #":"6","replicate #":"2","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"24h_3",
"Factors":{"Time":"24","Sample source":"Human osteosarcoma U2-OS cells"},
"Additional sample data":{"Treatment":"Dim","RAW_FILE_NAME(Sample_ID)":"18_WT_Dim_24h_3.mzML","condition #":"6","replicate #":"3","reference condition #":"1"}
},
{
"Subject ID":"-",
"Sample ID":"std003.mzML",
"Factors":{"Time":"-","Sample source":"standard"},
"Additional sample data":{"Treatment":"-","RAW_FILE_NAME(Sample_ID)":"std003.mzML","condition #":"-","replicate #":"-","reference condition #":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DoubleBlank.mzML",
"Factors":{"Time":"-","Sample source":"Blank"},
"Additional sample data":{"Treatment":"-","RAW_FILE_NAME(Sample_ID)":"DoubleBlank.mzML","condition #":"-","replicate #":"-","reference condition #":"-"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"The parental U2OS line was obtained from ATCC and engineered to express the Dmr-PERK construct in house.","SAMPLE_TYPE":"human osteosarcoma cell line","COLLECTION_METHOD":"Liquid Liquid Extraction- Matyash","COLLECTION_LOCATION":"Calico Life Sciences","COLLECTION_FREQUENCY":"1","COLLECTION_DURATION":"30 mins","VOLUMEORAMOUNT_COLLECTED":"0.8 mL","STORAGE_CONDITIONS":"-80℃","COLLECTION_VIALS":"2 mL glass vial","STORAGE_VIALS":"2 mL glass vial","COLLECTION_TUBE_TEMP":"4C","ADDITIVES":"none","TISSUE_CELL_IDENTIFICATION":"human osteosarcoma cell line"},

"TREATMENT":{"TREATMENT_SUMMARY":"U2OS cells were treated with 0.05nM of dimerizer over the course of 0,1,2,4,8,24 hours for lipidomics analysis","TREATMENT_COMPOUND":"DImerizer AP20187","TREATMENT_DOSE":"0.05 nM to media","TREATMENT_DOSEDURATION":"0h,1h,2h,4h,8h,24h","TREATMENT_VEHICLE":"ethanol","CELL_GROWTH_CONTAINER":"6-well plate","CELL_GROWTH_CONFIG":"U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1X antibiotic-antimycotic solution (Gibco). Cells were housed in an incubator at 37°C, 5% CO2, 20% O2 and passaged every 2-3 days with trypsin.","CELL_MEDIA":"U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1X antibiotic-antimycotic solution (Gibco).","CELL_HARVESTING":"LLE-MTBE (Matyash)","CELL_PCT_CONFLUENCE":"80%","CELL_MEDIA_LASTCHANGED":"24 hours prior to harvesting"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cells were cultured in 6-well plates, and ~500,000 cells per well were washed with warm PBS and quenched with 0.8 mL of 50/50 MeOH/H2O containing 100 µL of SPLASH LIPIDOMIXLipidSplash internal standards (Avanti Polar Lipids), followed by incubation at -20℃ for 15 minutes. Samples were scraped and collected to a 2 mL glass vial and extracted for lipidomics analysis using an MTBE-LLE extraction adopted from Matyash, et al 87. In brief, 800 μL of MTBE was added to the mixture, vortexed for 30 seconds and incubated on ice for another 15 minutes and centrifuged at 3500 RPM for 10 minutes at 4℃. Lipids partitioned in the top layer were collected into a separate vial, and the extraction process was repeated with an addition of 600 µL of MTBE. After incubating and centrifuging, the second organic layer was collected and combined in the first lipid vial, dried under nitrogen at 4℃, resuspended in 200 µL of ButOH/MeOH/H2O (2:1:1, v/v/v) for lipid analysis.","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACTION_METHOD":"Matyash","EXTRACT_STORAGE":"4℃","SAMPLE_RESUSPENSION":"ButOH/MeOH/H2O (2:1:1, v/v/v)"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"RPLC with a Thermo Accucore C30 for lipidomics analysis","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Thermo Scientific Accucore C30 (250 × 2.1mm, 2.6um, 150 Å)","SOLVENT_A":"60% acetonitrile/40% water; 20mM ammonium formate; 0.25μM medronic acid","SOLVENT_B":"90% isopropanol/10% acetonitrile; 20mM ammonium formate; 0.25μM medronic acid","FLOW_GRADIENT":"The gradient was t(min) = −7, 30% B, t = 0, 30% B, t = 7, 43% B, t = 12, 65% B, t = 30, 70% B, t = 31, 88% B, t = 51, 95% B, t = 53, 100% B, t = 55, 100% B, t = 55.1, 30% B, t = 60, 30% B.","FLOW_RATE":"200uL/min","COLUMN_TEMPERATURE":"35","COLUMN_PRESSURE":"900 bar","INJECTION_TEMPERATURE":"4C","INTERNAL_STANDARD":"Splash Lipidomix (Avanti Polar Lipids)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"The mass spectrometer settings were as follows: data-dependent acquisition (DDA) was performed with the following parameters: resolution = 140,000, AGC target = 3.00 × 106, maximum IT (ms) = 100, scan range = 200–2000. The MS2 parameters were as follows: resolution = 17,500, AGC target = 3.00 × 106, maximum IT (ms) = 150, loop count = 8, isolation window (m/z) = 1, (N)CE = 20, 30, 40; underfill ratio = 1.00%, Apex trigger(s) = 5–30, dynamic exclusion(s) = 15 s. Maven software was used for data processing (Seitzer, P., Bennett, B. & Melamud, E. MAVEN2: An Updated Open-Source Mass Spectrometry Exploration Platform. Metabolites 12, 684 (2022).) Analysis of the lipidomics and metabolomics data was performed using Calico Lipidomics and Metabolomics Analysis (clamanR) package (https://github.com/calico/claman) in R. Raw files were converted to the mxML format using ProteoWizard Ver 3 88. Compound identifications were detected and grouped using the OpenCLaM R package (https://github.com/calico/open_clam) and manually inspected using the MAVEN2 peak analysis program (https://github.com/eugenemel/maven) 85 with the criteria of a precursor ion tolerance of 10 ppm and a product ion tolerance of 20 ppm, comparing fragmentation and retention time to an in-house library generated from authentic standards for metabolomics, and in-house generated in-silico libraries for lipidomics (https://github.com/calico/CalicoLipidLibrary). Per feature, our software assigned a unique groupID in a numerical format."},

"MS_METABOLITE_DATA":{
"Units":"Log2(Top Peak Area)",

"Data":[{"Metabolite":"Ceramide(15:1;O2/15:0)","0h_1":"22.07677923","4h_1":"22.34709619","4h_2":"21.87844341","4h_3":"21.88009775","8h_1":"22.1383054","8h_2":"22.14993407","8h_3":"21.83326507","24h_1":"22.13788609","24h_2":"22.01822073","24h_3":"21.88329278","0h_2":"22.0484122","0h_3":"22.01898439","1h_1":"21.92158247","1h_2":"21.9355576","1h_3":"21.71778173","2h_1":"22.34372398","2h_2":"21.92043391","2h_3":"21.91022284"},{"Metabolite":"Ceramide(16:1;O2/18:1)","0h_1":"19.87997159","4h_1":"20.35849493","4h_2":"20.50831405","4h_3":"20.51071407","8h_1":"20.60826012","8h_2":"20.61584749","8h_3":"20.25903808","24h_1":"20.13117292","24h_2":"20.16237888","24h_3":"20.41388148","0h_2":"19.69219011","0h_3":"19.86946055","1h_1":"20.06650263","1h_2":"19.97228753","1h_3":"19.70878469","2h_1":"20.47250703","2h_2":"19.89746928","2h_3":"20.09022051"},{"Metabolite":"Ceramide(16:1;O2/24:0)","0h_1":"18.23531016","4h_1":"18.72524819","4h_2":"19.25943453","4h_3":"18.61622171","8h_1":"19.35807917","8h_2":"19.24998593","8h_3":"18.45771991","24h_1":"18.15332875","24h_2":"18.91147949","24h_3":"19.07893861","0h_2":"19.15019554","0h_3":"18.11038454","1h_1":"19.02864112","1h_2":"19.80755274","1h_3":"18.83305771","2h_1":"20.07765009","2h_2":"19.8410317","2h_3":"18.77118527"},{"Metabolite":"Ceramide(17:1;O2/24:0)","0h_1":"18.95703374","4h_1":"19.05965935","4h_2":"19.30551559","4h_3":"19.23850023","8h_1":"19.21310645","8h_2":"19.20358235","8h_3":"19.15687697","24h_1":"18.83162202","24h_2":"18.9850573","24h_3":"18.92330708","0h_2":"19.14972418","0h_3":"18.96862461","1h_1":"19.10288318","1h_2":"19.1162989","1h_3":"19.04626624","2h_1":"19.28301924","2h_2":"19.15815817","2h_3":"19.22459276"},{"Metabolite":"Ceramide(18:0;O2/16:0)","0h_1":"19.21419845","4h_1":"19.58794195","4h_2":"19.33013087","4h_3":"19.60519724","8h_1":"19.42212548","8h_2":"19.32600904","8h_3":"19.04827915","24h_1":"19.05518064","24h_2":"18.95253518","24h_3":"19.48978244","0h_2":"19.29726615","0h_3":"19.04738572","1h_1":"19.48526071","1h_2":"19.45663779","1h_3":"19.40946254","2h_1":"19.32091855","2h_2":"19.08802619","2h_3":"19.24309537"},{"Metabolite":"Ceramide(18:0;O2/24:0)","0h_1":"19.28772126","4h_1":"19.49399858","4h_2":"19.48385968","4h_3":"19.51570501","8h_1":"19.28855422","8h_2":"19.35111083","8h_3":"18.96536896","24h_1":"19.00569461","24h_2":"19.18672029","24h_3":"19.3053092","0h_2":"19.17516423","0h_3":"19.08668098","1h_1":"19.54986831","1h_2":"19.56844291","1h_3":"19.45139715","2h_1":"19.51282677","2h_2":"19.45259304","2h_3":"19.40312375"},{"Metabolite":"Ceramide(18:0;O2/24:1) (1)","0h_1":"17.84088864","4h_1":"17.89458419","4h_2":"18.07246761","4h_3":"18.21261913","8h_1":"17.55218419","8h_2":"17.90798821","8h_3":"17.37717835","24h_1":"17.46924173","24h_2":"17.59947976","24h_3":"17.73592111","0h_2":"17.93715288","0h_3":"17.92180708","1h_1":"18.34861849","1h_2":"18.24157866","1h_3":"18.22822596","2h_1":"17.97656797","2h_2":"17.8685189","2h_3":"17.84345004"},{"Metabolite":"Ceramide(18:0;O2/24:1) 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