{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002199","ANALYSIS_ID":"AN003599","VERSION":"1","CREATED_ON":"June 15, 2022, 5:34 pm"},

"PROJECT":{"PROJECT_TITLE":"FOXA2 controls the anti-oxidant response in FH-deficient cells independent of NRF2","PROJECT_SUMMARY":"Hereditary Leiomyomatosis and renal cell cancer is caused by fumarate hydratase loss of heterozygosity and subsequence accumulation of fumarate. Fumarate is known to activate the anti-oxidant response and is key for cellular survival. Fumarate succinates KEAP1 which releases NRF2 to activate the antioxidant response. The role of fumarate on the global regulatory chromatin landscape is less understood. Here, by integrating chromatin accessibility and histone ChIP-seq profiles, we identify complex transcription factor networks involved in the highly remodelled chromatin landscape of FH-deficient cells. We implicate FOXA2 in the maintenance of FH-deficient cells by regulating anti-oxidant response genes and subsequent metabolic output, independent of NRF2. These results identify new redox and amino acid metabolism regulators and provide new avenues for therapeutic intervention.","INSTITUTE":"CECAD Research Center","LAST_NAME":"Yang","FIRST_NAME":"Ming","ADDRESS":"Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany","EMAIL":"ming.yang@uni-koeln.de","PHONE":"4922147884306"},

"STUDY":{"STUDY_TITLE":"The metabolomics effect of Foxa2 knockdown on FH-deficient cells","STUDY_SUMMARY":"To assess whether there is a functional convergence between the two proteins, we performed metabolomics on Fh1fl/fl, Fh1-/-CL1 and Fh1-/-CL19 cells treated with either siNT or siFoxa2. 5 x 104 Fh1fl/fl, 1 x 105 Fh1-/-CL1 and 1 x 105 Fh1-/-CL19 cells were plated the onto 6-well plate and reverse transfected with siRNA. Before extraction, cells were counted using a separate counting plate. There are a total of 30 samples prepared from three cell lines and 6 biological conditions.","INSTITUTE":"CECAD Research Center","LAST_NAME":"Yang","FIRST_NAME":"Ming","ADDRESS":"Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany","EMAIL":"ming.yang@uni-koeln.de","PHONE":"4922147884306"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"CR02_031",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_031"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_032",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_032"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_033",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_033"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_034",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_034"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_035",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_035"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_036",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_036"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_037",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_037"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_038",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_038"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_039",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_039"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_040",
"Factors":{"Genotype":"Fh1fl/fl","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_040"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_041",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_041"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_042",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_042"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_043",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_043"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_044",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_044"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_045",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_045"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_046",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_046"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_047",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_047"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_048",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_048"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_049",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_049"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_050",
"Factors":{"Genotype":"Fh1-/-CL1","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_050"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_051",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_051"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_052",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_052"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_053",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_053"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_054",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_054"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_055",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siNT"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_055"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_056",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_056"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_057",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_057"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_058",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_058"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_059",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_059"}
},
{
"Subject ID":"-",
"Sample ID":"CR02_060",
"Factors":{"Genotype":"Fh1-/-CL19","Treatment":"siFoxa2"},
"Additional sample data":{"RAW_FILE_NAME":"CR02_060"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"5 x 104 Fh1fl/fl and 1 x 105 Fh1-/-CL1 and Fh1-/-CL19 cells were plated the onto 6-well plate and reverse transfected with siRNA. Before extraction, cells were counted using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept on cold bath with dry ice and methanol. Metabolite extraction buffer (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well following the proportion 1×106 cells/0.5 ml of buffer. Plates were kept at −80°C and kept overnight. The following day, the extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 10 min at 4°C, the supernatants were transferred into LC-MS vials.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"5 x 104 Fh1fl/fl and 1 x 105 Fh1-/-CL1 and Fh1-/-CL19 cells were reverse transfected in 6-well plates with 50 pmol of either control siNT (Horizon, D-001810-10-05) or siFoxa2 (Horizon, L-043601-00-0005) siRNA using 2.5 µl RNAiMAX per well. Cells were incubated for 72 hours before either RNA or protein were extracted."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"5 x 104 Fh1fl/fl and 1 x 105 Fh1-/-CL1 and Fh1-/-CL19 cells were plated the onto 6-well plate and reverse transfected with siRNA. Before extraction, cells were counted using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept on cold bath with dry ice and methanol. Metabolite extraction buffer (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well following the proportion 1×106 cells/0.5 ml of buffer. Plates were kept at −80°C and kept overnight. The following day, the extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 10 min at 4°C, the supernatants were transferred into LC-MS vials."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. Samples were randomized and the injection volume was 5 µl. A pooled quality control (QC) sample was generated from an equal mixture of all individual samples and analysed interspersed at regular intervals.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish Horizon","COLUMN_NAME":"SeQuant ZIC-pHILIC"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Exploris 240","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"Metabolites were measured with Vanquish Horizon UHPLC coupled to an Orbitrap Exploris 240 mass spectrometer (both Thermo Fisher Scientific) via a heated electrospray ionization source. The spray voltages were set to +3.5kV/-2.8 kV, RF lens value at 70, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The flow rate for sheath gas, aux gas and sweep gas were set to 40, 15 and 0, respectively. For MS1 scans, mass range was set to m/z=70-900, AGC target set to standard and maximum injection time (IT) set to auto. Data acquisition for experimental samples used full scan mode with polarity switching at an Orbitrap resolution of 120000. Data acquisition for untargeted metabolite identification was performed using the AcquireX Deep Scan workflow, an iterative data-dependent acquisition (DDA) strategy using multiple injections of the pooled sample. In brief, sample was first injected in full scan-only mode in single polarity to create an automated inclusion list. MS2 acquisition was then carried out in triplicate, where ions on the inclusion list were prioritized for fragmentation in each run, after which both the exclusion and inclusion lists were updated in a manner where fragmented ions from the inclusion list were moved to exclusion list for the next run. DDA full scan-ddMS2 method for AcquireX workflow used the following parameters: full scan resolution was set to 60000, fragmentation resolution to 30000, fragmentation intensity threshold to 5.0e3. Dynamic exclusion was enabled after 1 time and exclusion duration was 10s. Mass tolerance was set to 5ppm. Isolation window was set to 1.2 m/z. Normalized HCD collision energies were set to stepped mode with values at 30, 50, 150. Fragmentation scan range was set to auto, AGC target at standard and max IT at auto. Xcalibur AcquireX method modification was on. Mild trapping was enabled. Metabolite identification was performed in the Compound Discoverer software (v 3.2, Thermo Fisher Scientific). Metabolites were annotated at the MS2 level using both an in-house mzVault spectral database curated from 1051 authentic compound standards and the online spectral library mzCloud. The precursor mass tolerance was set to 5 ppm and fragment mass tolerance set to 10 ppm. Only metabolites with mzVault or mzCloud best match score above 50% and 75%, respectively, and RT tolerance within 0.5 min to that of a purified standard run with the same chromatographic method were exported to generate a list including compound names, molecular formula and RT. The curated list was then used for further processing in the Tracefinder software (v 5.0, Thermo Fisher Scientific), where extracted ion chromatographs for all compound were examined and manually integrated if necessary. False positive, noise or chromatographically unresolved compounds were removed. The peak area for each detected metabolite was then normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling through instrument analysis. The normalized areas were used as variables for further statistical data analysis."},

"MS_METABOLITE_DATA":{
"Units":"peak area",

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}

}