#METABOLOMICS WORKBENCH AlmaVS_20240925_060946 DATATRACK_ID:5225 STUDY_ID:ST003516 ANALYSIS_ID:AN005773 PROJECT_ID:PR002161 VERSION 1 CREATED_ON October 8, 2024, 3:43 pm #PROJECT PR:PROJECT_TITLE Macrophages are activated towards phagocytic Lymphoma Cell Clearance by Pentose PR:PROJECT_TITLE Phosphate Pathway Inhibition PR:PROJECT_TYPE LC-MS/MS targeted analysis PR:PROJECT_SUMMARY Macrophages in the B-cell lymphoma microenvironment represent a functional node PR:PROJECT_SUMMARY in progression and therapeutic response. We assessed metabolic regulation of PR:PROJECT_SUMMARY macrophages in the context of therapeutic antibody-mediated phagocytosis. PR:PROJECT_SUMMARY Pentose phosphate pathway (PPP) inhibition induced increased phagocytic lymphoma PR:PROJECT_SUMMARY cell clearance by macrophages in vitro, in primary human Chronic lymphocytic PR:PROJECT_SUMMARY leukemia (CLL) patient co-cultures, and in mouse models. Addition of the PPP PR:PROJECT_SUMMARY inhibitor S3 to antibody therapy achieved significantly prolonged overall PR:PROJECT_SUMMARY survival in an aggressive B-cell lymphoma mouse model. PPP inhibition induced PR:PROJECT_SUMMARY metabolic activation and pro-inflammatory polarization of macrophages while it PR:PROJECT_SUMMARY decreased macrophages´ support for survival of lymphoma cells empowering PR:PROJECT_SUMMARY anti-lymphoma function. As mechanism of macrophage repolarization, the link PR:PROJECT_SUMMARY between PPP and immune regulation was identified. PPP inhibition causes PR:PROJECT_SUMMARY decreased glycogen level and subsequent modulation of the immune modulatory PR:PROJECT_SUMMARY UDPG-Stat1-Irg1-Itaconate axis. Thus, we hypothesize the PPP as key regulator PR:PROJECT_SUMMARY and targetable modulator of macrophage activity in lymphoma to improve efficacy PR:PROJECT_SUMMARY of immunotherapies and prolong survival. PR:INSTITUTE Universidad CEU San Pablo PR:DEPARTMENT Química y Bioquímica PR:LAST_NAME Villaseñor PR:FIRST_NAME Alma PR:ADDRESS Urbanización Montepríncipe, n/s, Boadilla del Monte, Madrid, 28003, Spain PR:EMAIL alma.villasenor@ceu.es PR:PHONE 913724750 #STUDY ST:STUDY_TITLE Macrophages are activated towards phagocytic Lymphoma Cell Clearance by Pentose ST:STUDY_TITLE Phosphate Pathway Inhibition ST:STUDY_SUMMARY Macrophages in the B-cell lymphoma microenvironment represent a functional node ST:STUDY_SUMMARY in progression and therapeutic response. We assessed metabolic regulation of ST:STUDY_SUMMARY macrophages in the context of therapeutic antibody-mediated phagocytosis. ST:STUDY_SUMMARY Pentose phosphate pathway (PPP) inhibition induced increased phagocytic lymphoma ST:STUDY_SUMMARY cell clearance by macrophages in vitro, in primary human Chronic lymphocytic ST:STUDY_SUMMARY leukemia (CLL) patient co-cultures, and in mouse models. Addition of the PPP ST:STUDY_SUMMARY inhibitor S3 to antibody therapy achieved significantly prolonged overall ST:STUDY_SUMMARY survival in an aggressive B-cell lymphoma mouse model. PPP inhibition induced ST:STUDY_SUMMARY metabolic activation and pro-inflammatory polarization of macrophages while it ST:STUDY_SUMMARY decreased macrophages´ support for survival of lymphoma cells empowering ST:STUDY_SUMMARY anti-lymphoma function. As mechanism of macrophage repolarization, the link ST:STUDY_SUMMARY between PPP and immune regulation was identified. PPP inhibition causes ST:STUDY_SUMMARY decreased glycogen level and subsequent modulation of the immune modulatory ST:STUDY_SUMMARY UDPG-Stat1-Irg1-Itaconate axis. Thus, we hypothesize the PPP as key regulator ST:STUDY_SUMMARY and targetable modulator of macrophage activity in lymphoma to improve efficacy ST:STUDY_SUMMARY of immunotherapies and prolong survival. ST:INSTITUTE Universidad CEU San Pablo ST:DEPARTMENT Química y Bioquímica ST:LAST_NAME Villaseñor ST:FIRST_NAME Alma ST:ADDRESS Urbanización Montepríncipe, n/s, Boadilla del Monte, Madrid, 28003, Spain ST:EMAIL alma.villasenor@ceu.es ST:PHONE 913724750 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:CELL_STRAIN_DETAILS J774A.1 macrophages SU:CELL_COUNTS 3.94 · 10^6 #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 CTRL1 CTRL1 Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype RAW_FILE_NAME=CTRL1.mzML SUBJECT_SAMPLE_FACTORS CTRL2 CTRL2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype RAW_FILE_NAME=CTRL2.mzML SUBJECT_SAMPLE_FACTORS CTRL3 CTRL3 Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype RAW_FILE_NAME=CTRL3.mzML SUBJECT_SAMPLE_FACTORS CTRL4 CTRL4 Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype RAW_FILE_NAME=CTRL4.mzML SUBJECT_SAMPLE_FACTORS CTRL5 CTRL5 Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype RAW_FILE_NAME=CTRL5.mzML SUBJECT_SAMPLE_FACTORS OXY1 OXY1 Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=OXY1.mzML SUBJECT_SAMPLE_FACTORS OXY2 OXY2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=OXY2.mzML SUBJECT_SAMPLE_FACTORS OXY3 OXY3 Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=OXY3.mzML SUBJECT_SAMPLE_FACTORS OXY4 OXY4 Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=OXY4.mzML SUBJECT_SAMPLE_FACTORS OXY5 OXY5 Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=OXY5.mzML SUBJECT_SAMPLE_FACTORS PHY1 PHY1 Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=PHY1.mzML SUBJECT_SAMPLE_FACTORS PHY2 PHY2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=PHY2.mzML SUBJECT_SAMPLE_FACTORS PHY3 PHY3 Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=PHY3.mzML SUBJECT_SAMPLE_FACTORS PHY4 PHY4 Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=PHY4.mzML SUBJECT_SAMPLE_FACTORS PHY5 PHY5 Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=PHY5.mzML SUBJECT_SAMPLE_FACTORS ANA1 ANA1 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=ANA1.mzML SUBJECT_SAMPLE_FACTORS ANA2 ANA2 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=ANA2.mzML SUBJECT_SAMPLE_FACTORS ANA3 ANA3 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=ANA3.mzML SUBJECT_SAMPLE_FACTORS ANA4 ANA4 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=ANA4.mzML SUBJECT_SAMPLE_FACTORS ANA5 ANA5 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype RAW_FILE_NAME=ANA5.mzML SUBJECT_SAMPLE_FACTORS MLP1 MLP1 Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP RAW_FILE_NAME=MLP1.mzML SUBJECT_SAMPLE_FACTORS MLP2 MLP2 Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP RAW_FILE_NAME=MLP2.mzML SUBJECT_SAMPLE_FACTORS MLP3 MLP3 Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP RAW_FILE_NAME=MLP3.mzML SUBJECT_SAMPLE_FACTORS MLP4 MLP4 Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP RAW_FILE_NAME=MLP4.mzML SUBJECT_SAMPLE_FACTORS MLP5 MLP5 Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP RAW_FILE_NAME=MLP5.mzML SUBJECT_SAMPLE_FACTORS GPGD1 GPGD1 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 RAW_FILE_NAME=GPGD1.mzML SUBJECT_SAMPLE_FACTORS GPGD2 GPGD2 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 RAW_FILE_NAME=GPGD2.mzML SUBJECT_SAMPLE_FACTORS GPGD3 GPGD3 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 RAW_FILE_NAME=GPGD3.mzML SUBJECT_SAMPLE_FACTORS GPGD4 GPGD4 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 RAW_FILE_NAME=GPGD4.mzML SUBJECT_SAMPLE_FACTORS GPGD5 GPGD5 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 RAW_FILE_NAME=GPGD5.mzML SUBJECT_SAMPLE_FACTORS TKT1 TKT1 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 RAW_FILE_NAME=TKT1.mzML SUBJECT_SAMPLE_FACTORS TKT2 TKT2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 RAW_FILE_NAME=TKT2.mzML SUBJECT_SAMPLE_FACTORS TKT3 TKT3 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 RAW_FILE_NAME=TKT3.mzML SUBJECT_SAMPLE_FACTORS TKT4 TKT4 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 RAW_FILE_NAME=TKT4.mzML SUBJECT_SAMPLE_FACTORS TKT5 TKT5 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 RAW_FILE_NAME=TKT5.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L1 Cal_Curve_L1_rA Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L1_rA.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L1 Cal_Curve_L1_rB Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L1_rB.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L1 Cal_Curve_L1_rC Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L1_rC.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L2 Cal_Curve_L2_rA Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L2_rA.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L2 Cal_Curve_L2_rB Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L2_rB.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L2 Cal_Curve_L2_rC Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L2_rC.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L3 Cal_Curve_L3_rA Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L3_rA.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L3 Cal_Curve_L3_rB Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L3_rB.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L3 Cal_Curve_L3_rC Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L3_rC.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L4 Cal_Curve_L4_rA Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L4_rA.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L4 Cal_Curve_L4_rB Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L4_rB.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L4 Cal_Curve_L4_rC Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L4_rC.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L5 Cal_Curve_L5_rA Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L5_rA.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L5 Cal_Curve_L5_rB Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L5_rB.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L5 Cal_Curve_L5_rC Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L5_rC.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L6 Cal_Curve_L6_rA Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L6_rA.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L6 Cal_Curve_L6_rB Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L6_rB.mzML SUBJECT_SAMPLE_FACTORS Cal_Curve_L6 Cal_Curve_L6_rC Sample source:J774A.1_murine_macrophages | Experimental_Group:Calibration_Curve | Treatment:- | Genotype:ISTD+SAMPLE_MIX RAW_FILE_NAME=Cal_Curve_L6_rC.mzML SUBJECT_SAMPLE_FACTORS Blank_1 Blank_1 Sample source:- | Experimental_Group:Blank | Treatment:- | Genotype:Blank RAW_FILE_NAME=Blank_1.mzML SUBJECT_SAMPLE_FACTORS Blank_2 Blank_2 Sample source:- | Experimental_Group:Blank | Treatment:- | Genotype:Blank RAW_FILE_NAME=Blank_2.mzML SUBJECT_SAMPLE_FACTORS Blank_3 Blank_3 Sample source:- | Experimental_Group:Blank | Treatment:- | Genotype:Blank RAW_FILE_NAME=Blank_3.mzML SUBJECT_SAMPLE_FACTORS Blank_4 Blank_4 Sample source:- | Experimental_Group:Blank | Treatment:- | Genotype:Blank RAW_FILE_NAME=Blank_4.mzML #COLLECTION CO:COLLECTION_SUMMARY J774A.1 macrophages were from ATCC and DSMZ. All cell lines were cultured on CO:COLLECTION_SUMMARY 6-well plates or 10cm dishes from Corning and incubated with 5% CO2 at 37,0 °C. CO:COLLECTION_SUMMARY J774A.1 cells were cultured in DMEM containing 10% fetal bovine serum and 1% CO:COLLECTION_SUMMARY penicillin/streptomycin. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY 1x107 J774A.1 (wildtype, empty vector control, shRNA 6PGD knockdown or shRNA TKT TR:TREATMENT_SUMMARY knockdown) in 10mL media were plated out on 10cm dishes and incubated for 24hrs. TR:TREATMENT_SUMMARY J774A.1 wildtype dishes were treated with PPP inhibitors. The cells were TR:TREATMENT_SUMMARY incubated for 24hrs. Cells were scraped, washed with DPBS and resuspended in 1mL TR:TREATMENT_SUMMARY DPBS. The cells were counted using CASY cell counter and analyser. Cells were TR:TREATMENT_SUMMARY centrifuged at 300g for 5min and supernatant was discarded. Cell pellet was TR:TREATMENT_SUMMARY crash frozen using liquid nitrogen. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Macrophages pellets were resuspended in 50 uL of cold methanol (MeOH, -20ºC) SP:SAMPLEPREP_SUMMARY containing labelled standard mixture, vortex-mixed for 2 minutes and incubated SP:SAMPLEPREP_SUMMARY on ice for 5 minutes in order to precipitate the protein content. The labelled SP:SAMPLEPREP_SUMMARY standard mixture was composed of 2-Ketoglutaric acid D6, Succinic acid D6, and SP:SAMPLEPREP_SUMMARY Glutathione -glycine-13C2,15N trifluoro. After this step, samples were sonicated SP:SAMPLEPREP_SUMMARY for 4 minutes to break the cells, and 50 uL of water was added to extract and SP:SAMPLEPREP_SUMMARY solubilize the metabolites. Samples were again vortex-mixed for 2 minutes, and SP:SAMPLEPREP_SUMMARY centrifuged for 20 minutes at 4ºC, at 13200 rpm. 60 uL of the supernatant was SP:SAMPLEPREP_SUMMARY transferred to liquid chromatography vials to analysis. 20 uL of the remaining SP:SAMPLEPREP_SUMMARY supernatant of each sample were taken to make a pool for the quality control SP:SAMPLEPREP_SUMMARY sample (QC) and built a standard addition calibration curve. Samples and the SP:SAMPLEPREP_SUMMARY calibration curve standards were analysed using liquid chromatography coupled to SP:SAMPLEPREP_SUMMARY mass spectrometry with a triple cuadrupole mass analyser (LC-QqQ-MS) in tandem SP:SAMPLEPREP_SUMMARY analysis mode. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Method for the analysis of metabolites derived from the pentose phosphate CH:CHROMATOGRAPHY_SUMMARY pathway CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Agilent ZORBAX RRHD SB-C18 (150 x 2.1mm,1.8um) CH:SOLVENT_A 97% water/3% methanol; 10mM tributylamine; 15mM acetic acid CH:SOLVENT_B 100% methanol; 10mM tributylamine; 15mM acetic acid CH:FLOW_GRADIENT 0% B (0.00-2.50 min), 0-20% B (2.50-7.50 min), 20-45% B (7.50-13.00 min), 45-99% CH:FLOW_GRADIENT B (13.00-20.00 min) and 99% B (20.00-24.00 min) CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 35ºC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The LC system was coupled to an Agilent 6460 triple quadrupole mass spectrometer MS:MS_COMMENTS using an ESI interface working in multiple reaction monitoring (MRM) mode MS:MS_COMMENTS (Agilent Technologies, Waldbronn, Germany). Metabolites were ionized in an ESI MS:MS_COMMENTS source operating in negative ionization mode. The drying gas flow rate was 13 MS:MS_COMMENTS L/min at 150 °C, and the nebulizer was set to 45 psi. The sheath gas flow rate MS:MS_COMMENTS was set to 12 L/min at 350 °C; capillary and nozzle voltages were set to 2000 MS:MS_COMMENTS and 500 V, respectively. Data were acquired in dynamic multiple reaction MS:MS_COMMENTS monitoring 503 mode, using a cycle time of 650 ms. Output raw data files were MS:MS_COMMENTS preprocessed with Agilent MassHunter Workstation Software Quantitative Analysis MS:MS_COMMENTS for QQQ, from which a metabolite matrix containing the integrated area and MS:MS_COMMENTS retention time (RT) for specific compounds was obtained. The concentration of MS:MS_COMMENTS the samples were calculated by interpolation in calibration curves, and MS:MS_COMMENTS normalized by the number of cells. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS uM MS_METABOLITE_DATA_START Samples CTRL1 CTRL2 CTRL3 CTRL4 CTRL5 OXY1 OXY2 OXY3 OXY4 OXY5 PHY1 PHY2 PHY3 PHY4 PHY5 ANA1 ANA2 ANA3 ANA4 ANA5 MLP1 MLP2 MLP3 MLP4 MLP5 GPGD1 GPGD2 GPGD3 GPGD4 GPGD5 TKT1 TKT2 TKT3 TKT4 TKT5 Factors Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Control | Treatment:- | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Oxythiamine | Treatment:Oxythiamine_520µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:Physcion | Treatment:Physcion_9µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:6-aminonicotinamide | Treatment:6-aminonicotinamide_11µM_for_24hrs | Genotype:wildtype Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP Sample source:J774A.1_murine_macrophages | Experimental_Group:empty_vector_control | Treatment:- | Genotype:MLP Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 Sample source:J774A.1_murine_macrophages | Experimental_Group:6-phosphogluconate_dehydrogenase_knockdown | Treatment:- | Genotype:sh6PGD2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 Sample source:J774A.1_murine_macrophages | Experimental_Group:Transketolase_knockdown | Treatment:- | Genotype:shTKT2 Adenosine_5-diphosphate 18.45 12.20 15.47 13.06 11.60 12.79 13.59 17.93 14.82 17.49 24.37 18.60 20.90 26.54 29.92 18.89 28.52 29.34 27.79 12.20 20.16 21.03 27.92 21.44 29.40 20.07 26.61 6.78 24.15 25.74 29.50 30.62 42.97 49.47 59.50 Adenosine_5-triphosphate 1.74 1.47 1.36 1.82 1.01 2.03 1.60 2.04 1.89 2.26 3.52 2.24 2.35 3.87 4.12 6.96 5.58 5.55 6.70 6.48 6.03 10.72 9.94 9.51 5.12 10.84 13.38 31.44 13.02 6.77 5.32 3.75 16.71 10.57 34.78 Lactic_acid 60.96 41.95 57.10 26.98 43.32 24.37 33.08 34.68 40.33 41.76 21.54 37.32 37.86 54.48 72.71 34.07 87.18 95.64 55.21 10.75 17.68 33.17 25.30 26.60 46.06 46.81 62.04 63.74 63.02 68.00 98.31 107.77 113.87 103.46 67.69 Pyruvic_acid 6.74 2.87 8.33 3.19 5.85 8.06 7.12 7.33 7.05 8.69 4.60 6.11 7.90 7.77 8.67 6.80 8.80 6.62 5.93 4.56 4.60 7.04 5.08 5.02 6.87 18.07 18.99 50.10 17.99 11.11 12.03 16.09 15.15 16.73 9.40 alpha-Ketoglutaric_acid 0.09 0.08 0.12 0.11 0.10 0.10 0.12 0.07 0.12 0.10 0.17 0.10 0.19 0.16 0.20 0.09 0.13 0.11 0.09 0.14 0.15 0.16 0.16 0.14 0.15 0.23 0.30 0.25 0.20 0.21 0.29 0.43 0.25 0.39 0.49 NAD 3.44 2.68 2.90 2.51 2.48 2.83 3.41 2.72 2.97 3.92 3.59 3.59 3.23 4.61 4.84 3.16 4.66 4.87 3.96 3.72 3.94 3.90 4.79 3.33 3.59 1.36 2.44 1.28 1.95 3.54 4.41 5.36 5.38 5.36 5.06 Fructose_6-phosphate 2.95 2.34 2.91 1.95 2.17 2.08 2.44 2.54 2.17 2.77 3.72 2.87 2.34 3.07 4.08 5.96 6.11 6.15 8.34 6.51 8.44 5.49 9.76 6.84 5.16 2.98 3.31 1.57 3.33 5.22 7.51 8.84 8.18 9.12 6.24 Glucose_6-phosphate 1.57 1.44 1.75 1.51 1.09 1.20 1.28 1.19 1.05 1.34 2.76 1.56 1.35 1.55 1.82 8.54 5.61 7.39 10.66 6.56 8.04 2.26 9.29 6.23 2.19 0.33 0.43 0.51 0.46 1.68 2.63 2.32 1.79 1.93 2.03 Ribose/Ribulose_5-phosphate 2.06 2.12 2.02 2.76 1.52 2.40 1.92 1.84 1.79 2.89 4.68 1.96 1.77 2.25 2.94 7.25 6.55 11.34 13.54 16.85 4.29 5.82 4.57 4.87 2.40 0.33 0.34 0.22 0.38 0.31 4.59 4.04 5.02 3.74 6.48 Xylulose/Ribose/Ribulose_5-phosphate 8.63 9.05 5.75 8.02 3.31 3.97 2.91 4.76 2.66 4.73 11.72 3.21 3.07 3.14 5.64 4.35 6.03 9.89 15.58 15.32 13.93 5.48 15.44 8.98 7.07 1.25 1.67 2.77 1.96 1.60 5.74 2.95 3.80 3.22 7.80 Sedoheptulose_7-phosphate 12.07 11.38 16.31 10.50 8.94 16.63 15.34 11.75 11.77 15.70 18.67 12.33 10.40 11.35 18.49 28.85 20.12 27.29 32.83 19.54 18.32 18.74 18.44 16.35 11.26 3.22 4.68 2.32 5.16 7.49 9.16 11.63 9.77 10.14 6.64 Glutathione_Reduced 579.19 425.33 568.81 490.91 418.48 420.76 458.32 433.20 425.15 620.27 654.73 599.17 521.47 598.39 840.74 554.44 706.74 762.14 591.62 506.34 696.54 648.58 689.24 524.04 649.16 92.16 143.47 171.61 151.62 223.16 631.00 628.21 494.64 655.54 482.02 NADP 4.07 2.84 3.64 2.64 2.77 2.68 2.96 2.78 2.94 3.77 4.59 3.97 3.76 3.57 4.94 1.60 3.31 3.40 3.19 1.74 3.83 2.90 4.86 2.92 3.57 2.09 3.00 2.52 2.54 4.02 6.99 6.81 6.20 6.59 5.47 Succinic_acid 10.04 10.85 5.38 9.27 4.67 3.87 4.70 5.13 4.90 5.71 12.04 9.94 7.70 11.45 14.45 3.51 6.56 9.13 7.96 3.88 6.89 7.51 10.03 6.24 6.65 2.37 3.98 4.68 3.88 3.95 7.64 6.29 7.09 6.62 16.37 Itaconic_acid 5.24 3.83 4.58 2.58 4.01 2.02 2.14 2.26 2.56 2.88 5.82 7.38 6.00 7.14 8.05 1.70 3.66 3.46 3.24 1.53 1.73 1.44 2.69 1.40 2.85 0.17 0.24 0.17 0.22 0.36 1.09 1.21 1.11 1.11 1.01 L-Malic_acid 11.67 11.81 10.07 11.31 8.14 6.56 8.88 7.14 7.16 8.62 13.18 11.41 8.14 12.66 15.74 6.97 14.51 16.29 13.43 5.84 8.77 9.20 12.09 7.64 9.34 21.43 27.75 21.28 23.40 23.65 14.61 16.87 13.58 15.21 19.28 Citric_acid 2.33 1.70 2.21 2.00 1.76 1.94 1.82 1.77 2.21 2.27 2.77 3.32 3.85 4.01 4.09 4.09 6.61 4.18 3.35 2.37 4.18 5.14 4.20 3.78 2.73 0.38 0.87 0.53 0.90 1.56 2.30 2.73 3.92 4.30 3.80 NADH 0.06 0.01 0.03 0.02 0.04 0.02 0.04 0.05 0.22 0.09 0.01 0.16 0.01 0.05 0.04 0.06 0.02 0.09 0.10 0.11 0.02 0.02 0.02 0.09 0.02 0.12 0.13 0.12 0.03 0.02 0.04 0.02 0.03 0.04 0.02 DL-Glyceraldehyde_3-phosphate_(area_norm) 34704.16 19739.26 33352.33 24093.26 22726.91 27960.63 30338.40 34872.01 26828.63 39283.18 36873.72 36846.79 28057.41 34123.61 48405.25 24297.26 41297.28 33517.07 55812.83 29024.78 31129.16 36676.95 40508.77 27294.30 35992.48 18945.03 25304.54 20319.62 21194.44 29902.53 51887.04 50459.44 47841.44 51742.87 31815.55 "D-Fructose_1,6-biphosphate_(area_norm)" 835.69 669.74 951.46 343.84 692.18 807.24 786.51 756.20 879.67 930.06 702.54 1330.27 861.14 1346.65 1479.86 2623.71 3626.79 3558.86 2043.70 672.43 622.29 2540.48 697.68 1039.47 1348.26 912.25 2030.15 1870.64 4294.31 10142.86 3341.94 3338.34 2870.76 3346.07 1715.18 6-Phosphogluconic_acid_(area_norm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 189479.19 259663.36 290334.65 85419.54 126768.31 0.00 0.00 0.00 5686.78 0.00 4627.93 6002.35 7901.63 7041.72 0.00 0.00 0.00 0.00 0.00 1506.76 Erytrose-4-phosphate_(area_norm) 120092.58 122459.27 174846.69 114299.41 96562.28 182646.38 159516.25 124864.51 122813.91 169883.10 210157.68 128524.07 114750.53 121596.54 206355.04 385741.22 278261.06 342936.35 439761.73 270683.55 249700.24 220253.38 244532.28 221708.87 123785.21 27557.33 40557.61 16264.54 39475.06 48625.07 60889.24 91778.42 88125.09 82566.31 57791.67 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChemidentifiers Precursor ion m/z Product ion m/z Adenosine_5-diphosphate 6022 426 328 Adenosine_5-triphosphate 5957 506 408.1 Lactic_acid 612 89 43.3 Pyruvic_acid 1060 87 43.2 alpha-Ketoglutaric_acid 51 145 101 NAD 5892 662.1 540 Fructose_6-phosphate 69507 259 97 Glucose_6-phosphate 5958 259 79 Ribose/Ribulose_5-phosphate 10975657/439184 229 79 Xylulose/Ribose/Ribulose_5-phosphate 5289590/10975657/439184 229 79 Sedoheptulose_7-phosphate 165007 289 96.9 Glutathione_Reduced 124886 306.3 143 NADP 5885 741.7 619.9 Succinic_acid 1110 117 73.1 Itaconic_acid 811 129 85.1 L-Malic_acid 222656 133 115 Citric_acid 311 191 111 NADH 439153 664.4 78.9 DL-Glyceraldehyde_3-phosphate_(area_norm) 729 169 97 "D-Fructose_1,6-biphosphate_(area_norm)" 10267 338.9 97 6-Phosphogluconic_acid_(area_norm) 91493 275.1 78.9 Erytrose-4-phosphate_(area_norm) - 199 97 METABOLITES_END #END