#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