#METABOLOMICS WORKBENCH kenanazam_20160908_171249_mwtab.txt DATATRACK_ID:729 STUDY_ID:ST000460 ANALYSIS_ID:AN000721 PROJECT_ID:PR000354 VERSION 1 CREATED_ON September 8, 2016, 5:22 pm #PROJECT PR:PROJECT_TITLE Effect of ATG7 reconstitution in AGY571 cells PR:PROJECT_TYPE Amino acid steady-state level analysis PR:PROJECT_SUMMARY AGY571 is a B cell lymphoma isolated from Eu-Myc CD19-Cre;ATG7fl/fl mice. AGY571 PR:PROJECT_SUMMARY cells engineered to express rtTA and Dox-inducible GFP or ATG7 were treated with PR:PROJECT_SUMMARY 500 ng/mL or 1 ug/mL Doxycycline for 48 hours. Assessing the effects of ATG7 PR:PROJECT_SUMMARY reexpression in B cell lymphoma. PR:INSTITUTE H. Lee Moffitt Cancer Center PR:DEPARTMENT Tumor Biology PR:LABORATORY John Cleveland PR:LAST_NAME Fernandez PR:FIRST_NAME Mario PR:ADDRESS 12902 Magnolia Drive, MRC 4-West PR:EMAIL mario.fernandez@moffitt.org PR:PHONE 813-745-5140 PR:FUNDING_SOURCE 5R01CA154739 #STUDY ST:STUDY_TITLE Effect of ATG7 reconstitution in AGY571 cells ST:STUDY_TYPE Cells are treated with vehicle or Doxycycline - to induce expression of GFP or ST:STUDY_TYPE ATG7 ST:STUDY_SUMMARY AGY571 cells engineered to express Dox-inducible GFP or ATG7 were treated with ST:STUDY_SUMMARY 500 ng/mL or 1 ug/mL Doxycycline for 48 hours. Assessing the effects of ATG7 ST:STUDY_SUMMARY reexpression in B cell lymphoma. ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LABORATORY John Cleveland ST:LAST_NAME Fernandez ST:FIRST_NAME Mario ST:ADDRESS 12902 Magnolia Drive, MRC 4-West ST:EMAIL mario.fernandez@moffitt.org ST:PHONE 813-745-5140 ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 15 ST:STUDY_COMMENTS Groups collected in triplicate per time point, either treated or untreated with ST:STUDY_COMMENTS Doxycycline. #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:CELL_STRAIN_DETAILS AGY571 SU:SUBJECT_COMMENTS See study design section for cell number per sample for cell counts SU:CELL_PRIMARY_IMMORTALIZED Immortalized #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS AGY571_GFP AGY571_001 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:GFP (ATG7-/-) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=9 SUBJECT_SAMPLE_FACTORS AGY571_GFP AGY571_002 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:GFP (ATG7-/-) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=9.36 SUBJECT_SAMPLE_FACTORS AGY571_GFP AGY571_003 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:GFP (ATG7-/-) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=6.705 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_004 Treatment:Vehicle | Dox Concentration:0 ug/mL | Ectopic Gene Expression:None (ATG7-/- backgound) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=8.91 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_005 Treatment:Vehicle | Dox Concentration:0 ug/mL | Ectopic Gene Expression:None (ATG7-/- backgound) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=4.725 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_006 Treatment:Vehicle | Dox Concentration:0 ug/mL | Ectopic Gene Expression:None (ATG7-/- backgound) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=7.74 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_007 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:ATG7 Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=7.74 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_008 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:ATG7 Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=4.68 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_009 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:ATG7 Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=5.31 SUBJECT_SAMPLE_FACTORS AGY571_GFP AGY571_010 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:GFP (ATG7-/-) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=7.584 SUBJECT_SAMPLE_FACTORS AGY571_GFP AGY571_011 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:GFP (ATG7-/-) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=7.056 SUBJECT_SAMPLE_FACTORS AGY571_GFP AGY571_012 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:GFP (ATG7-/-) Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=5.16 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_013 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:ATG7 Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=8.496 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_014 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:ATG7 Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=7.728 SUBJECT_SAMPLE_FACTORS AGY571_ATG7 AGY571_015 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:ATG7 Hours=48; Genotype=Eu-Myc ATG7-/-; Cell Count (in millions)=9.264 #COLLECTION CO:COLLECTION_SUMMARY Cells were counted, cells were collected via centrifugation, washed twice with CO:COLLECTION_SUMMARY cold 1x PBS, PBS removed and flash frozen. Pellets were saved at -80 C. CO:SAMPLE_TYPE Cell CO:COLLECTION_METHOD Pellet CO:COLLECTION_LOCATION Lab CO:COLLECTION_FREQUENCY 48 hours after Dox treatment CO:STORAGE_CONDITIONS Pellet at -80C CO:STORAGE_VIALS 1.5 mL microfuge tube CO:TISSUE_CELL_IDENTIFICATION AGY571 cells #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with vehicle (dH2O) or 500 ng/mL doxycycline. TR:TREATMENT_COMPOUND Doxycycline TR:TREATMENT_DOSE 0.5-1.0 ug/mL TR:TREATMENT_DOSEVOLUME 5 mL TR:TREATMENT_DOSEDURATION 48 hours TR:TREATMENT_VEHICLE dH2O TR:CELL_GROWTH_CONTAINER 6-well plate #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Amino acid SP:SAMPLEPREP_PROTOCOL_FILENAME SB_AA_Assay.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Amino Acid CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters AccQ Tag (100 x 2mm, 1.7um) CH:METHODS_FILENAME SB_AA_Assay.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Sanford Burnham #MS MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Amino Acids #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/mg protein MS_METABOLITE_DATA_START Samples AGY571_004 AGY571_005 AGY571_006 AGY571_001 AGY571_002 AGY571_003 AGY571_010 AGY571_011 AGY571_012 AGY571_007 AGY571_008 AGY571_009 AGY571_013 AGY571_014 AGY571_015 Factors Treatment:Vehicle | Dox Concentration:0 ug/mL | Ectopic Gene Expression:None (ATG7-/- backgound) Treatment:Vehicle | Dox Concentration:0 ug/mL | Ectopic Gene Expression:None (ATG7-/- backgound) Treatment:Vehicle | Dox Concentration:0 ug/mL | Ectopic Gene Expression:None (ATG7-/- backgound) Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:GFP (ATG7-/-) Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:GFP (ATG7-/-) Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:GFP (ATG7-/-) Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:GFP (ATG7-/-) Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:GFP (ATG7-/-) Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:GFP (ATG7-/-) Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:ATG7 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:ATG7 Treatment:Doxycycline | Dox Concentration:1 ug/mL | Ectopic Gene Expression:ATG7 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:ATG7 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:ATG7 Treatment:Doxycycline | Dox Concentration:500 ng/mL | Ectopic Gene Expression:ATG7 Alanine 1.49 3.07 1.95 1.45 1.53 1.76 1.27 1.26 1.66 1.44 2.30 2.42 1.42 1.65 1.76 Arginine 0.10 0.18 0.15 0.12 0.08 0.10 0.06 0.10 0.12 0.09 0.18 0.17 0.09 0.10 0.08 Asparagine 0.12 0.24 0.14 0.12 0.10 0.17 0.11 0.12 0.15 0.10 0.18 0.18 0.10 0.11 0.10 Aspartate 0.52 1.03 0.60 0.64 0.47 0.78 1.22 1.15 1.61 0.54 0.93 0.91 0.65 0.86 0.77 Glutamate 4.62 9.31 5.55 4.18 4.36 5.80 4.91 6.32 7.98 4.96 9.86 8.52 4.64 5.79 5.78 Glutamine 3.76 8.56 6.03 4.60 4.20 5.85 5.19 5.72 7.48 4.51 5.96 7.83 4.41 4.44 4.69 Glycine 1.22 2.41 1.43 1.07 0.99 1.63 1.07 1.04 1.99 1.38 1.94 2.27 1.18 1.53 1.33 Histidine 0.04 0.11 0.08 0.05 0.07 0.09 0.06 0.06 0.11 0.04 0.07 0.10 0.06 0.07 0.07 Isoleucine 0.09 0.25 0.22 0.13 0.16 0.25 0.17 0.17 0.23 0.11 0.15 0.29 0.17 0.15 0.20 Leucine 0.11 0.29 0.19 0.14 0.16 0.22 0.14 0.17 0.25 0.12 0.18 0.23 0.15 0.17 0.18 Lysine 0.46 0.27 0.19 0.20 0.33 0.34 0.21 Phenylalanine 0.07 0.22 0.17 0.08 0.12 0.15 0.09 0.11 0.17 0.08 0.14 0.17 0.12 0.13 0.16 Proline 1.08 1.94 1.08 0.88 0.86 1.21 0.63 0.75 0.98 0.96 1.46 1.78 0.80 0.90 0.78 Serine 0.32 0.54 0.33 0.35 0.33 0.51 0.27 0.37 0.64 0.39 0.54 0.58 0.30 0.37 0.37 Threonine 0.87 1.82 1.05 0.90 0.82 0.84 0.86 0.91 1.26 0.85 1.52 1.60 0.82 0.94 0.77 Tryptophan 0.03 0.04 0.04 0.03 0.03 0.05 0.03 0.03 0.04 0.03 0.04 0.05 0.04 0.03 0.03 Tyrosine 0.10 0.32 0.20 0.14 0.17 0.22 0.14 0.17 0.27 0.12 0.18 0.25 0.16 0.18 0.17 Valine 0.39 0.28 0.18 0.22 0.30 0.21 0.29 0.36 0.22 0.23 0.23 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID Alanine 4.16 260.17 5950 Arginine 2.68 345.21 6322 Asparagine 2.25 303.13 6267 Aspartate 2.99 304.11 5960 Glutamate 3.35 318.11 33032 Glutamine 2.65 317.21 5961 Glycine 2.81 246.08 750 Histidine 2.07 326.21 6274 Isoleucine 8.15 302.21 6306 Leucine 8.26 302.21 6106 Lysine 6.66 487.21 5962 Phenylalanine 8.40 336.21 6140 Proline 4.84 286.16 145742 Serine 2.60 276.11 5951 Threonine 3.74 290.11 6288 Tryptophan 8.54 375.21 6305 Tyrosine 6.66 352.21 6057 Valine 7.09 288.21 6287 METABOLITES_END #END