#METABOLOMICS WORKBENCH Metabolomics4_20210625_064116 DATATRACK_ID:2718 STUDY_ID:ST001850 ANALYSIS_ID:AN002998 PROJECT_ID:PR001167 VERSION 1 CREATED_ON June 29, 2021, 5:37 am #PROJECT PR:PROJECT_TITLE NonTargeted LC-MS-based metabolomics analysis for both whole cell and PR:PROJECT_TITLE mitochondria metabolites to gain an insight into the role of Tug1/PGC1 axis on PR:PROJECT_TITLE metabolite profiles in podocytes PR:PROJECT_TYPE Biomarker PR:PROJECT_SUMMARY We found that double mutant Tug1-KD/Pgc1-OE rescued the effects of Tug1-KD on PR:PROJECT_SUMMARY basal, maximal, and spare capacity respiration rates in podocytes. Therefore we PR:PROJECT_SUMMARY employed an unbiased LCMS based metabolomics analysis or both whole cell and PR:PROJECT_SUMMARY mitochondria metabolites to gain an insight into the role of Tug1/PGC1 on PR:PROJECT_SUMMARY metabolite profiles in podocytes. PR:INSTITUTE University of Texas MD Anderson Cancer Center PR:DEPARTMENT Nephrology PR:LAST_NAME Danesh PR:FIRST_NAME Farhad PR:ADDRESS 1515 Holcombe Blvd, Houston, TX77030 PR:EMAIL fdanesh@mdanderson.org PR:PHONE 7135634498 #STUDY ST:STUDY_TITLE Unbiased LC-MS-based metabolomics analysis for both whole cell and mitochondria ST:STUDY_TITLE metabolites to gain an insight into the role of Tug1/PGC1 axis on metabolite ST:STUDY_TITLE profiles in podocytes ST:STUDY_SUMMARY unbiased LC-MS-based metabolomics analysis for both whole cell and mitochondria ST:STUDY_SUMMARY metabolites to gain an insight into the role of Tug1/PGC1 axis on metabolite ST:STUDY_SUMMARY profiles in podocytes ST:INSTITUTE University of Texas MD Anderson Cancer Center ST:LAST_NAME Danesh ST:FIRST_NAME Farhad ST:ADDRESS 1515 Holcombe Blvd, Houston ,TX77030 ST:EMAIL fdanesh@mdanderson.org ST:PHONE 7135634498 ST:NUM_GROUPS 3 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - PLKO_1_Hilic Treatment:PLKO RAW_FILE_NAME=210111_AA_PLKO_1 SUBJECT_SAMPLE_FACTORS - PLKO_2_Hilic Treatment:PLKO RAW_FILE_NAME=210111_AA_PLKO_2 SUBJECT_SAMPLE_FACTORS - PLKO_3_Hilic Treatment:PLKO RAW_FILE_NAME=210111_AA_PLKO_3 SUBJECT_SAMPLE_FACTORS - Tugl-KD_1_Hilic Treatment:Tug1-KD RAW_FILE_NAME=210111_AA_Tugl-KD_1 SUBJECT_SAMPLE_FACTORS - Tugl-KD_2_Hilic Treatment:Tug1-KD RAW_FILE_NAME=210111_AA_Tugl-KD_2 SUBJECT_SAMPLE_FACTORS - Tugl-KD_3_Hilic Treatment:Tug1-KD RAW_FILE_NAME=210111_AA_Tugl-KD_3 SUBJECT_SAMPLE_FACTORS - Tugl-KD_PGCOE_1_Hilic Treatment:PGCOE RAW_FILE_NAME=210111_AA_Tugl-KD_PGCOE_1 SUBJECT_SAMPLE_FACTORS - Tugl-KD_PGCOE_2_Hilic Treatment:PGCOE RAW_FILE_NAME=210111_AA_Tugl-KD_PGCOE_2 SUBJECT_SAMPLE_FACTORS - Tugl-KD_PGCOE_3_Hilic Treatment:PGCOE RAW_FILE_NAME=210111_AA_Tugl-KD_PGCOE_3 SUBJECT_SAMPLE_FACTORS - PLKO_1_IC Treatment:PLKO RAW_FILE_NAME=210111_Cells_PLKO_1 SUBJECT_SAMPLE_FACTORS - PLKO_2_IC Treatment:PLKO RAW_FILE_NAME=210111_Cells_PLKO_2 SUBJECT_SAMPLE_FACTORS - PLKO_3_IC Treatment:PLKO RAW_FILE_NAME=210111_Cells_PLKO_3 SUBJECT_SAMPLE_FACTORS - Tugl-KD_1_IC Treatment:Tug1-KD RAW_FILE_NAME=210111_Cells_Tug1-KD_1 SUBJECT_SAMPLE_FACTORS - Tugl-KD_2_IC Treatment:Tug1-KD RAW_FILE_NAME=210111_Cells_Tug1-KD_2 SUBJECT_SAMPLE_FACTORS - Tugl-KD_3_IC Treatment:Tug1-KD RAW_FILE_NAME=210111_Cells_Tug1-KD_3 SUBJECT_SAMPLE_FACTORS - Tugl-KD_PGCOE_1_IC Treatment:PGCOE RAW_FILE_NAME=210111_Clees_Tug1-KD_PGCOE_1 SUBJECT_SAMPLE_FACTORS - Tugl-KD_PGCOE_2_IC Treatment:PGCOE RAW_FILE_NAME=210111_Clees_Tug1-KD_PGCOE_2 SUBJECT_SAMPLE_FACTORS - Tugl-KD_PGCOE_3_IC Treatment:PGCOE RAW_FILE_NAME=210111_Clees_Tug1-KD_PGCOE_3 #COLLECTION CO:COLLECTION_SUMMARY Briefly, podocytes were cultured on BD BioCoat Collagen I plates (BD CO:COLLECTION_SUMMARY Biosciences, San Jose, CA) at 33°C in RPMI 1640 complete media with 20 U/ml CO:COLLECTION_SUMMARY mouse recombinant IFN-g (Thermo Fisher, Carlsbad, CA). To induce CO:COLLECTION_SUMMARY differentiation, we cultured podocytes in DMEM (5.5mM glucose and 5% FBS) at CO:COLLECTION_SUMMARY 37°C without IFN-g for 10-12 days. To rescue the expression of PGC1-a in stable CO:COLLECTION_SUMMARY Tug1-knockdown CRISPR clone (Tug1-KD)(Long et al., 2016), CMV CO:COLLECTION_SUMMARY enhancer/promoter-driven mouse Pgc1-a cDNA (Addgene, Watertown, MA) was inserted CO:COLLECTION_SUMMARY into vector Zeo-pT-MCS-GFP-T2A-Puro(Long et al., 2020), a modified PiggyBac CO:COLLECTION_SUMMARY transposon system, selected with 1ug/ml puromycin and sorted by GFP, to generate CO:COLLECTION_SUMMARY Tug1-KD/Pgc1-OE. We isolated kidney podocytes by positive selection with CO:COLLECTION_SUMMARY biotin-labelled Kirrel3 and Podocalyxin antibodies (2.5 μg/antibody/mouse, R&D CO:COLLECTION_SUMMARY Systems, Minneapolis, MN) followed by Streptavidin M-280 Dynabeads as previously CO:COLLECTION_SUMMARY described (Badal et al., 2016). CO:SAMPLE_TYPE Epithelial cells #TREATMENT TR:TREATMENT_SUMMARY we cultured podocytes in DMEM (5.5mM glucose and 5% FBS) at 37°C without IFN-g TR:TREATMENT_SUMMARY for 10-12 days. To rescue the expression of PGC1-a in stable Tug1-knockdown TR:TREATMENT_SUMMARY CRISPR clone (Tug1-KD)(Long et al., 2016), CMV enhancer/promoter-driven mouse TR:TREATMENT_SUMMARY Pgc1-a cDNA (Addgene, Watertown, MA) was inserted into vector TR:TREATMENT_SUMMARY Zeo-pT-MCS-GFP-T2A-Puro(Long et al., 2020), a modified PiggyBac transposon TR:TREATMENT_SUMMARY system, selected with 1ug/ml puromycin and sorted by GFP, to generate TR:TREATMENT_SUMMARY Tug1-KD/Pgc1-OE. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from cell samples (in triplicates) grown on 10cm dishes were SP:SAMPLEPREP_SUMMARY extracted with ice-cold 80% methanol. After centrifugation, extracts in SP:SAMPLEPREP_SUMMARY supernatants were dried by evaporation under nitrogen. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY liquid chromatography (LC)-MS CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters XBridge Amide (100 x 4.6mm, 3.5um) #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME MD Anderson Metabolomics Core AN:OPERATOR_NAME Lin Tan #MS MS:INSTRUMENT_NAME Thermo Fusion Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data were acquired using a Thermo Orbitrap Fusion Tribrid Mass Spectrometer MS:MS_COMMENTS under ESI positive ionization mode at a resolution of 240,000.Raw data files MS:MS_COMMENTS were imported to Thermo Trace Finder software for final analysis. The relative MS:MS_COMMENTS abundance of each metabolite was normalized by DNA concentrations. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC/ngDNA MS_METABOLITE_DATA_START Samples PLKO_1_Hilic PLKO_2_Hilic PLKO_3_Hilic Tugl-KD_1_Hilic Tugl-KD_2_Hilic Tugl-KD_3_Hilic Tugl-KD_PGCOE_1_Hilic Tugl-KD_PGCOE_2_Hilic Tugl-KD_PGCOE_3_Hilic Factors Treatment:PLKO Treatment:PLKO Treatment:PLKO Treatment:Tug1-KD Treatment:Tug1-KD Treatment:Tug1-KD Treatment:PGCOE Treatment:PGCOE Treatment:PGCOE 4-hydroxyproline 2.93e-03 2.88e-03 2.66e-03 2.16e-03 2.39e-03 2.77e-03 2.51e-03 2.14e-03 2.62e-03 Acetylcholine 2.88e-04 2.50e-04 2.42e-04 4.46e-04 2.77e-04 2.03e-04 8.81e-05 1.37e-04 1.32e-04 Alanine 1.39e-02 1.51e-02 1.55e-02 1.17e-02 8.87e-03 9.77e-03 1.41e-02 1.24e-02 1.53e-02 Arginine 1.27e-02 1.11e-02 1.31e-02 1.18e-02 1.41e-02 1.41e-02 1.87e-02 2.13e-02 1.47e-02 Asparagine 2.19e-03 2.39e-03 2.39e-03 1.67e-03 1.57e-03 1.44e-03 2.95e-03 1.92e-03 3.49e-03 Aspartate 2.00e-03 2.17e-03 2.49e-03 3.03e-03 2.58e-03 2.27e-03 1.89e-03 2.24e-03 2.92e-03 Carnitine 6.48e-02 5.59e-02 6.42e-02 8.76e-02 7.44e-02 7.57e-02 2.34e-02 2.09e-02 2.82e-02 Citrulline 4.18e-04 3.99e-04 2.62e-04 3.68e-03 3.69e-03 4.43e-03 3.27e-04 3.30e-04 3.13e-04 Creatine 4.01e-03 5.54e-03 5.78e-03 2.91e-03 2.54e-03 5.27e-03 4.02e-03 3.00e-03 6.21e-03 Creatinine 2.00e-02 1.74e-02 1.81e-02 1.26e-02 1.43e-02 1.80e-02 1.60e-02 1.78e-02 1.44e-02 Glutamate 2.86e-01 3.26e-01 3.26e-01 3.63e-01 3.13e-01 3.14e-01 2.87e-01 2.57e-01 3.25e-01 Glutamine 9.93e-02 1.07e-01 1.11e-01 1.21e-01 1.02e-01 9.66e-02 7.79e-02 8.62e-02 9.27e-02 Glycine 1.30e-04 1.91e-04 1.90e-04 8.50e-05 1.00e-04 1.36e-04 1.33e-04 1.55e-04 1.72e-04 Histidine 5.74e-03 5.47e-03 5.75e-03 5.71e-03 6.40e-03 6.83e-03 7.51e-03 7.78e-03 6.74e-03 hypotaurine 1.17e-02 1.25e-02 1.27e-02 1.26e-02 1.11e-02 1.16e-02 7.94e-03 6.88e-03 9.46e-03 IsoLeucine 7.70e-02 6.14e-02 6.29e-02 6.14e-02 7.46e-02 7.29e-02 9.03e-02 9.43e-02 7.27e-02 Leucine 7.06e-02 5.99e-02 5.74e-02 5.66e-02 7.27e-02 6.88e-02 8.10e-02 8.77e-02 6.85e-02 Lysine 8.44e-03 6.72e-03 7.66e-03 7.99e-03 9.72e-03 1.10e-02 1.27e-02 1.38e-02 9.90e-03 Methionine 1.60e-02 1.43e-02 1.35e-02 1.20e-02 1.75e-02 1.68e-02 1.87e-02 2.35e-02 1.79e-02 Ornithine 1.53e-04 1.59e-04 1.15e-04 5.79e-04 6.84e-04 7.82e-04 2.54e-04 2.92e-04 2.63e-04 phenylalaine 6.60e-02 5.44e-02 5.23e-02 4.72e-02 7.64e-02 7.08e-02 8.42e-02 9.26e-02 6.88e-02 Proline 1.11e-01 1.24e-01 1.12e-01 5.72e-02 6.65e-02 7.15e-02 1.15e-01 1.09e-01 1.18e-01 pyroglutamic acid 5.73e-04 3.86e-04 3.87e-04 4.75e-04 4.49e-04 4.26e-04 4.62e-04 6.59e-04 4.16e-04 Sarcosine 6.01e-03 7.16e-03 7.66e-03 8.41e-03 7.50e-03 7.09e-03 4.71e-03 5.27e-03 6.36e-03 Serine 2.91e-03 3.30e-03 3.41e-03 3.61e-03 3.14e-03 3.08e-03 2.51e-03 2.61e-03 3.06e-03 Threonine 3.29e-02 3.44e-02 3.28e-02 2.85e-02 2.54e-02 2.68e-02 3.10e-02 2.51e-02 2.93e-02 Tryptophan 1.30e-02 9.75e-03 9.24e-03 8.32e-03 1.43e-02 1.36e-02 1.51e-02 1.64e-02 1.15e-02 Tyrosine 2.58e-02 2.13e-02 2.25e-02 2.75e-02 2.99e-02 2.92e-02 2.74e-02 3.46e-02 2.70e-02 Valine 4.34e-02 3.93e-02 3.70e-02 4.04e-02 4.38e-02 4.42e-02 5.23e-02 5.38e-02 4.38e-02 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 4-hydroxyproline Acetylcholine Alanine Arginine Asparagine Aspartate Carnitine Citrulline Creatine Creatinine Glutamate Glutamine Glycine Histidine hypotaurine IsoLeucine Leucine Lysine Methionine Ornithine phenylalaine Proline pyroglutamic acid Sarcosine Serine Threonine Tryptophan Tyrosine Valine METABOLITES_END #END