THE BOARD OF CONTROL FOR CRICKET IN INDIA, MUMBAI V. PR. CIT (CENTRAL) - 3 , MUMBAI

ITA 7202/MUM/2019

• Counselvise Citation: [2019] 112 COUNSELVISE.COM (IT) 4383 (ITAT-MUMBAI)
• Duration Of Justice: 4 day(s)
• Appeal Type: Income Tax Appeal
• Pronouncement Date: 26-11-2019
• Appeal Filed By: Assessee
• Order Result: Allowed
• Bench Allotted: B
• Assessment Year: 2015-2016
• Appeal Filed On: 22-11-2019
• Result in Favour of: Assessee
• Court: TRIBUNAL
• Section: 6; 2; 48; 13; 22; 20

Judgement Text

CHEMISTRY & BIOLOGY, VOLUME 22 SUPPLEMENTAL INFORMATION DIFFERENTIAL REGULATION OF SPECI FI C SPHINGOLIPIDS IN COLON CANCER CELLS DURING STAUROSPORINE-INDUCED APOPTOSIS VIRGINIA DEL SOLAR, DARLENY Y. LIZARDO, NASI LI, JEROD J. HURST, CHRISTOPHER J. BRAIS, AND G. EKIN ATILLA-GOKCUMEN S 1 SUPPLEMENTAL DATA FIGURE S1 . V IABILITY ASSAY FOR COLON CELL LINES AND PARP CLEAVAGE IN NON - CANCEROUS AND CANCEROUS CELL LINES ( RELATED TO FIGURE 1 A, FIGURE 5C AND SUPPLEMENTAL EXPERIMENTAL PROCEDURE CELL CULTURE AND TREATMENTS) . S2 FIGURE S2. PARP CLEAVAGE AND CERAMIDE/DIHYDROCERAMIDE FOLD CHANGES IN HCT - 116 CELLS AFTER DOXORUBICIN TREATMENT ( RELATED TO TABLE 1 ) . S3 FIGURE S3 . EFFECT OF STAUROSPORINE AND C16 - CERAMIDE COMBINATION TREATMENT ON VIABILITY OF HCT - 116 CELL S ( RELATED TO FIGURE 5C) . S4 TABLE S1 . MATRIX EFFECT AND QUANTIFICATION OF CERAMIDES IN HCT - 116 AND CCD - 112 CELLS (RELATED TO FIGURE 5A AND E XPERIMENTAL P ROCEDURE PREPARATION OF LIPID EXTRACTS, LC - MS METHOD AND DATA ANALYSIS AND S UPPLEMENTAL EXPERIMENTAL PROCEDURE NORMALIZATION AND THE MATRIX EFFECT AND QUANTIFICATION OF CERAMIDES BY LC - MS ). S5 TABLE S 2. TARGETED ANALYSIS OF LIPIDS DURING APOPTOSIS IN HCT - 116 CELLS ( RELATED TO FIGURE 2 ). S6 TABLE S3 . TARGETED ANALYSIS OF CERAMIDES, DIHYDROCERAMIDES, SPHINGOMYELINS AND DIHYDROSPHINGOMYELINS DURING APOPTOSIS IN NON - CANCEROUS (CCD - 112) AND CANCEROUS (HCT - 116) COLON CELL LINES (RELATED TO FIGURE 5 A AND FIGURE 5 B) . S7 TABLE S4 . TARGETED ANALYSIS OF CERAMIDES AND DIHYDROCERAMIDES RELEASED INTO THE CULTURE MEDIA DURING APOPTOSIS IN HCT - 116 ( RELATED TO FIGURE 3 AND SUPPLEMENTAL EXPERIMENTAL PROCEDURE MEDIA EXTRACTION) . S8 TABLE S5 . TARGETED ANALYSIS OF CERAMIDES AND DIHYDROCERAMIDES DURING APOPTOSIS IN NON - CANCEROUS (RPE - 1) AND CANCEROUS (HELA S3 AND MCF - 7) CELLS (RELAT ED TO FIGURE 5 A). S9 SUPPLEMENTAL EXPERIMENTAL PROCEDURES S10 - 11 SUPPLEMENTAL LIPID ASSIGNMENTS S12 - 15 SUPPLEMENTAL R EFERENCES S1 6 S 2 FIGURE S1 . VIABILITY ASSAY FOR COLON CELL LINES AND PARP CLEAVAGE IN NON - CANCEROUS AND CANCEROUS CELL LINES ( RELATED TO FIGURE 1 A , FIGURE 5C AND SUPPLEMENTAL E XPERIMENTAL P ROCEDURE CELL CULTURE AND TREATMENTS ) . (A) CELL VIABILITY IN THE PRESENCE OF DIFFERENT CONCENTRATIONS OF STAUROSPORINE IN HCT - 116 AND CCD - 112 COLON CELLS. DATA FROM THREE INDEPENDENT EXPERIMENTS (N = 15) ARE SHOWN AS MEAN SD. (B - C) WESTERN BLOTS OF CELL LINES TREATED FOR 5 - 24 HOURS WITH DMSO (0) AND STAUROSPORINE (STS) AT DIFFERENT CONCENTRATIONS. LYSATES WERE ANALYZED FOR PARP AND - TUBULIN. TWO BANDS AR E SHOWN IN PARP, FULL - LENGTH PARP (116 KDA) AND CLEAVED (CL) PARP (89 KDA). THE CLEAVED FORM IS A MARKER OF APOPTOSIS; - TUBULIN WAS USED AS LOADING CONTROL. (B) CANCER CELL LINES: HCT - 116, HUMAN COLORECTAL CARCINOMA; HELA S3, HUMAN CERVICAL CARCINOMA; MCF - 7, HUMAN BREAST ADENOCARCINOMA. (C) NON - CANCER CELL LINES: CCD - 112, HUMAN COLON FIBROBLAST ; RPE - 1, HUMAN RETINAL PIGMENTED EPITHELIUM. (D) WESTERN BLOT OF HCT - 116 CELL LINE TREATED WITH C16 - CERAMIDE (C16 CER) AT DIFFERENT CONCENTRATIONS DURING 16H. S 3 FIGURE S2. PARP CLEAVAGE AND CERAMIDE/DIHYDROCERAMIDE FOLD CHANGES IN HCT - 116 CELL S AFTER DOXORUBICIN TREATMENT ( RELATED TO TABLE 1 ) . (A) WESTERN BLOT OF HCT - 116 TREATED WITH DMSO (0) OR 2 M DOXORUBICIN FOR 48 H. LYSATES WERE ANALYZED FOR PARP AND - TUBULIN. TWO BANDS ARE SHOWN IN PARP, FULL - LENGTH PARP (116 KDA) AND CLEAVED (CL) PARP (89 KDA). THE CLEAVED FORM IS A MARKER OF APOPTOSIS; - TUBULIN WAS USED AS LOADING CONTROL. (B) FOLD CHANGES IN CERAMIDE AND DIHYDROCERAMIDE LEVELS IN HCT - 116 CELLS AFTER 2 M DOXORUBICIN TREATMENT FOR 48H. DOTTED LINE INDICATES NO CHANGE (FOLD CHANGE=1). DATA FROM TWO INDEPENDENT EXPERIMENTS (N = 10) ARE SHOWN AS MEAN SD. FOR EACH CERAMIDE AND DIHYDROCERAMIDE FOLD DIFFERENCE IS DETERMINED AS [ABUNDANCE 48H - TR EATMENT ] / [ABUNDANCE CONTROL ]. ABUNDANCE IS THE TOTAL ION COUNT FOR A GIVEN ION. LIPID COMPOSITION WAS NORMALIZED BASED ON PROTEIN CONCENTRATION AND INTERNAL STANDARDS. DOTTED LINE INDICATES EQUAL LEVELS IN BOTH TREATED AND NON - TREATED CELL S . S 4 FIGURE S3 . EFFECT OF STAUROSPORINE AND C16 - C ERAMIDE COMBINATION TREATMENT ON VIABILITY OF HCT - 116 CELL S ( RELATED TO FIGURE 5C ) . MTT ASSAY WAS PERFORMED TO DETERMINE THE VIABILITY OF HCT - 116 CELL LINE TREATED WITH EITHER STAUROSPORINE (0.1 M) OR C16 - CERAMIDE (0.1 M) OR IN COMBINATION OF BOTH COMPOUNDS FOR 16 HOURS. DATA FROM TWO INDEPENDENT EXPERIMENTS (N = 10) ARE SHOWN AS MEAN SD. S 5 TABLE S 1. MATRIX EFFECT AND QUANTIFICATION OF CERAMIDES IN HCT - 116 AND CCD - 112 CELLS. 13 C 18 - OLEIC ACID AND C17 - CERAMIDE WERE USED AS INTERNAL STANDARDS IN MATRIX EFFECT EVALUATION A (RELATED TO FIGURE 5A AND E XPERIMENTAL P ROCEDURE PREPARATION OF LIPID EXTRACTS, LC - MS METHOD AND DATA ANALYSIS AND S UPPLEMENTAL E XPERIMENTAL P ROCEDURE NORMALIZATION AND THE MATRIX EFFECT AND QUANTIFICATION OF CERAMIDES BY LC - MS ). AREA CCD - 112 HCT - 116 [NG/MG] CCD - 112 HCT - 116 RATIO F 13 C 18 - OLEIC ACID B M/ Z = 299.3090 E 6.52 0.56E+06 6.20 1.24E+06 C14 CER 1.34 0.01 1.44 0.17 0.93 C17 CER C M/Z = 550.5205 E 1.31 0.11E+07 1.55 0.17E+07 C16 CER 3.38 0.38 4.05 0.81 0.83 C14:0 FA D M/Z = 227.2017 E 1.33 0.05E+07 1.43 0.18E+07 C22 CER 0.30 0.02 0.18 0.03 1.67 C16:0 FA D M/Z = 255.2330 E 6.85 1.86E+07 5.73 0.94E+07 C24 CER 4.25 0.35 2.02 0.14 2.10 A DATA ARE GIVEN AS AVERAGE SD OF THREE INDEPENDENT EXPERIMENTS. B AREA WAS DETERMINED AS (ABUNDANCE) / [ 13 C 18 - OLEIC ACID ]. ABUNDANCE IS THE TOTAL ION COUNT FOR A GIVEN ION. EACH ION CORRESPONDS TO A MASS - TO - CHARGE RATIO ( M/Z ), WHICH IS USED TO ASSIGN THE SPECIE. C AREA WAS DETERMINED AS (ABUNDANCE) / [C17 CER]. D FA, FATTY ACID. AREA WA S DETERMINED AS (ABUNDANCE) OF SPECIFIC LIPID M/Z. E M/Z S CORRESPOND ING TO [M - H] - . NO SIGNIFICANT DIFFERENCES IN ABUNDANCES WERE OBSERVED DUE TO MATRIX EFFECT BETWEEN HCT - 116 AND CCD - 112 ( P 0.1) . F RATIO WAS DETERMINED AS [ NG/MG PROTEIN ] CCD - 112 / [ NG/MG PROTEIN ] HCT - 116 FOR EACH CERAMIDE. S 6 TABLE S 2 . TARGETED ANALYSIS OF LIPIDS DU RING APOPTOSIS IN HCT - 116 CELLS A ( RELATED TO FIGURE 2 ). LIPID FAMILY LIPID M/Z RT FC B (16H /C) LIPID M/Z RT FC ( 16H /C ) LIPID M/Z RT FC ( 16H /C ) FA C C14:0 227.2011 31.9 0.9 C18:1 281.2481 35.6 1.1 C24:0 367.3576 45.3 1.4 C16:0 255.2324 34.9 1.0 C18:2 279.2324 34.0 1.1 C24:1 365.3420 42.8 1.5 C16:1 253.2168 32.8 1.1 C20:0 311.2950 40.3 1.0 C26:0 395.3889 47.6 1.2 C18:0 283.2637 37.5 1.1 C20:4 303.2324 34.4 0.9 PA C LPA C16:0 409.2355 47.6 0.7 C34:1 673.4808 41.6 0.5 C36:2 699.4965 42.2 1.1 C32:0 647.4652 39.7 0.3 C34:2 671.4652 40.3 0.6 C36:3 701.5121 43.7 1.1 C32:1 645.4495 41.0 0.4 C36:1 697.4808 41.1 0.8 C38:4 723.4965 43.0 1.5 PE C LPE C18:0 480.3090 39.9 0.5 LPE C20:4 500.2777 36.1 0.6 C36:2 742.5387 52.9 0.8 LPE C18:1 478.2934 37.7 0.7 C36:1 744.5543 54.5 0.6 C38:4 766.5387 53.6 0.5 PS C LPS C18:0 524.2988 32.1 1.0 C34:1 760.5129 42.7 1.2 C36:4 782.4972 42.7 1.1 LPS C18:1 522.2832 30.3 1.2 C34:2 758.4972 41.3 1.8 C38:4 810.5285 44.0 2.5 C32:0 734.4972 42.0 0.8 C36:1 788.5442 44.7 1.1 C38:5 808.5129 43.1 1.5 C32:1 732.4816 40.7 1.3 C36:2 786.5285 43.1 1.6 PI C LPI C18:0 599.3196 37.7 1.5 C34:1 835.5337 49.2 1.1 C36:4 857.5180 48.3 1.1 LPI C18:1 597.3040 35.6 1.6 C34:2 833.5180 47.7 1.4 C38:4 885.5493 50.5 1.2 C32:0 809.5180 48.7 0.7 C36:1 863.5650 51.2 1.5 C38:5 883.5337 48.8 1.3 C32:1 807.5024 47.3 1.1 C36:2 861.5493 49.7 1.3 ST D CHOLESTEROL 369.3521 67.0 1.0 PC E LPC C16:0 496.3403 44.6 1.0 C34:1 760.5856 57.9 0.9 C36:3 784.5856 57.6 0.9 LPC C18:0 524.3716 47.3 1.1 C34:2 758.5700 57.1 1.0 C36:4 782.5700 57.3 0.9 LPC C18:1 522.3560 45.5 1.3 C34:3 756.5543 56.2 1.1 C38:4 810.6013 58.7 1.0 C32:0 734.5700 57.3 0.7 C36:1 788.6169 59.2 1.0 C38:5 808.5856 57.6 0.8 C32:1 732.5543 56.6 0.9 C36:2 786.6013 58.3 0.9 DG D,E C32:0 551.5039 57.9 0.3 C36:1 605.5509 59.6 0.9 C38:4 627.5352 59.0 1.2 C32:1 549.4883 56.2 0.6 C36:2 603.5352 58.7 0.9 C38:5 643.5302 61.0 0.6 C34:1 577.5196 58.4 0.6 C36:3 601.5196 57.2 2.1 C34:2 575.5039 57.6 1.1 C36:4 599.5039 56.9 1.1 TG F C50:1 850.7864 66.6 0.7 C54:3 902.8177 66.9 2.3 C56:2 932.8646 67.6 0.8 C52:2 876.8020 66.8 1.4 C54:4 900.8020 66.6 2.3 C56:6 924.8020 66.5 2.3 C54:2 904.8330 67.2 1.3 C54:6 896.7707 66.1 2.0 C56:8 920.7707 66.1 2.7 A ABBREVIATIONS: RT, RETENTION TIME; FC, FOLD CHANGE; 16H , 16H STAUROSPORINE - TREATED CELLS; C, CONTROL CELLS ; FA, FATTY ACID; PA, PHOSPHATIDIC ACID; PE, PHOSPHATIDYLETHANOLAMINE; PS, PHOSPHATIDYLSERINE; PI, PHOSPHATIDYLINOSITOL; ST, STEROL; PC, PHOSPHATIDYLCHOLINE; DG, DIACYLGLYCEROL; TG, TRIACYLGLYCEROL . B FC WAS DETERMINED AS [ABUNDAN CE 16H ] / [ABUNDANCE C] FOR EACH LIPID. ABUNDANCE IS THE TOTAL ION COUNT FOR A GIVEN ION. EACH ION CORRESPONDS TO A MASS - TO - CHARGE RATIO ( M/Z ), WHICH IS USED TO ASSIGN THE LIPID SPECIES. C M/Z S CORRESPOND ING TO [M - H] - . D M/Z S CORRESPOND ING TO [M +H - H 2 O ] + . E M/Z S CORRESPOND ING TO [M+H] + . F M/Z S CORRESPOND ING TO [M+NH 4 ] + . S 7 TABLE S 3 . TARGETED ANALYSIS OF CERAMIDES, DIHYDROCERAMIDES, SPHINGOMYELINS AND DIHYDROSPHINGOMYELINS DURING APOPTOSIS IN NON - CANCEROUS (CCD - 112) AND CANCEROUS (HCT - 116) COLON CELL LINES A - C (RELATED TO FIGURE 5 A AND FIGURE 5 B ) . CCD - 112 HCT - 116 ACYL CHAIN LIPID FC D (5H/C) FC D ( 16H /C ) L IPID FC (5H/C) FC ( 16H /C ) ACYL CHAIN LIPID FC (5H/C) FC ( 16H /C ) L IPID FC (5H/C) FC ( 16H /C ) C14 CER 0.8 1.3 SM 0.6 1.1 C14 CER 1.0 5.4 SM 1.2 3.0 DIHCER E - - DIHSM 1.1 1.6 DIHCER 6.2 12.4 DIHSM 2.5 6.1 C16 CER 0.6 1.3 SM 0.8 1.1 C16 CER 0.5 5.0 SM 1.1 1.5 DIHCER 2.4 4.0 DIHSM 1.6 2.2 DIHCER 3.0 11.9 DIHSM 2.4 4.4 C18 CER 0.9 1.2 SM 1.0 1.1 C18 CER 0.9 5.0 SM 1.5 2.4 DIHCER E - - DIHSM E - - DIHCER 5.8 13.8 DIHSM E - - C20 CER E - - SM E - - C20 CER 0.9 4.6 SM E - - DIHCER E - - DIHSM E - - DIHCER 4.2 8.5 DIHSM E - - C22 CER 0.6 1.0 SM 0.9 1.2 C22 CER 5.2 13.8 SM 1.5 3.3 DIHCER 1.8 2.8 DIHSM E - - DIHCER 2.0 3.0 DIHSM E - - C24 CER 0.7 1.0 SM 0.9 1.4 C24 CER 2.0 5.1 SM 1.3 3.5 DIHCER 1.3 2.1 DIHSM E - - DIHCER 1.2 2.0 DIHSM 1.3 3.9 C26 CER 1.5 2.2 SM 0.7 1.9 C26 CER 2.2 3.9 SM 1.1 4.7 DIHCER E - - DIHSM E - - DIHCER E - - DIHSM E - - A ABBREVIATIONS: FC, FOLD CHANGE; 5H, 5H STAUROSPORINE - TREATED CELLS ; 16H , 16H STAUROSPORINE - TREATED CELLS ; C, CONTROL CELLS . B CERAMIDES AND DIHYDROCERAMIDES: M/Z S CORRESPOND ING TO [M - H] - . SPHINGOMYELINS AND DIHYDROSPHING O MYELINS: M/Z S CORRESPONDING TO [M] + . C M/Z S AND THE CORRESPONDING RT FOR EACH SPECIES CAN BE FOUND IN THE S UPPLEMENTAL LIPID ASSIGNMENTS . D FC WA S DETERMINED AS [ABUNDANCE 16H OR 5 H] / [ABUNDANCE C] FOR EACH LIPID. ABUNDANCE IS THE TOTAL ION COUNT FOR A GIVEN ION. EACH ION CORRESPONDS TO A MASS - TO - CHARGE RATIO ( M/Z ), WHICH IS USED TO ASSIGN THE LIPID SPECIES. E NOT DETECTED. S 8 TABLE S 4. TARGETED ANALYSIS OF CERAMIDES AND DIHYDROCERAMIDES RELEASED INTO THE CULTURE ME DIA DURING APOPTOSIS IN HCT - 116 A,B ( RELATED TO FIGURE 3 AND S UPPLEMENTAL E XPERIMENTAL P ROCEDURE MEDIA EXTRACTION ) . L IPID OBSERVED M/Z RT FC ( 16H /C ) C L IPID OBSERVED M/Z RT FC ( 16H /C ) C14 CER 508.4818 62.2 ACCUM D C14 DIHCER 510.4923 62.8 ACCUM D C16 CER 536.5067 64.0 2.6 C16 DIHCER 538.5215 64.5 2.5 C18 CER E - - - C18 DIH CER E - - - C20 CER E - - - C20 DIH CER E - - - C22 CER 620.6046 68.1 1.1 C22 DIHCER 622.6160 68.5 1.0 C24 CER 648.6360 69.6 1.2 C24 DIHCER 650.6509 70.1 1.1 C26 CER E - - - C26 DIH CER E - - - A ABBREVIATIONS: RT, RETENTION TIME; FC, FOLD CHANGE; 16H , 16H STAUROSPORINE - TREATED CELLS; C, CONT ROL CELLS; ACCUM, ACCUMULATION . B M/Z S CORRESPONDING TO [M - H] - . C FC WAS DETERMINED AS [ABUNDANCE 16H ] / [ABUNDANCE C] FOR EACH LIPID. ABUNDANCE IS THE TOTAL ION COUNT FOR A GIVEN ION. EACH ION CORRESPONDS TO A MASS - TO - CHARGE RATIO ( M/Z ), WHICH IS USED TO ASSIGN THE LIPID SPECIES. D NOT DETECTED IN CONTROL. FOLD INCREASE COULD NOT BE DETERMINED DUE TO THE LOW ABUNDANCE IN CONTROL CELLS. E NOT DETECTED . S 9 TABLE S 5. TARGETED ANALYSIS OF CERAMIDES AND DIHYDROCERAMIDES DURING APOPTOSIS IN NON - CANCEROUS (RPE - 1) AND CANCEROUS (HELA S3 AND MCF - 7) CELLS A - C (RELATED TO FIGURE 5 A ). RPE - 1 HELA S3 MCF - 7 ACYL CHAIN LIPID FC D (5H/C) FC D ( 16H /C ) ACYL CHAIN LIPID FC (5H/C) FC ( 16H /C ) ACYL CHAIN LIPID FC (5H/C) FC ( 16H /C ) C14 CER 1.4 2.0 C14 CER 1.5 3.0 C14 CER 1.9 4.0 DIHCER 1.3 1.4 DIHCER 3.0 7.4 DIHCER 5.6 19.5 C16 CER 1.4 1.6 C16 CER 1.1 2.4 C16 CER 1.4 3.8 DIHCER 2.4 1.6 DIHCER 2.5 5.3 DIHCER 3.6 10.1 C18 CER 1.7 1.2 C18 CER 0.9 1.6 C18 CER 1.5 5.8 DIHCER 1.9 1.2 DIHCER 1.9 5.7 DIHCER 5.0 24.8 C20 CER 1.5 1.2 C20 CER 1.3 2 .6 C20 CER 1.8 7.6 DIHCER 1.3 1.1 DIHCER 1.4 2.8 DIHCER 5.0 24.7 C22 CER 1.5 1.5 C22 CER 1.5 3.1 C22 CER 1.3 3.5 DIHCER 2.1 1.3 DIHCER 2.1 5.0 DIHCER 3.0 7.4 C24 CER 1.5 2.5 C24 CER 1.3 3.7 C24 CER 2.1 5.2 DIHCER 1.9 1.9 DIHCER 1.8 4.1 DIHCER 1.6 2.7 C26 CER 2.0 3.0 C26 CER E - - C26 CER 0.9 1.7 DIHCER E - - DIHCER E - - DIHCER E - - A ABBREVIATIONS: FC, FOLD CHANGE; 5H, 5H STAUROSPORINE - TREATED CELLS ; 16H, 16H STAUROSPORINE - TREATED CELLS; C, CONTROL CELLS. B M/Z S CORRESPONDING TO [M - H] - . C M/Z S AND THE CORRESPONDING RT FOR EACH SPECIES CAN BE FOUND IN THE SUPPLEMENTAL EXPERIMENTAL PROCEDURE WITHIN LIPID IDENTIFICATION SECTION. D FC WAS DETERMINED AS [ABUNDANCE 16H OR 5 H] / [ABUNDANCE C] FOR EACH LIPID. ABUNDANCE IS THE TOTAL ION COUNT FOR A G IVEN ION. EACH ION CORRESPONDS TO A MASS - TO - CHARGE RATIO ( M/Z ), WHICH IS USED TO ASSIGN THE LIPID SPECIES. E NOT DETECTED. S 10 SUPLEMENTAL EXPERIMENTAL PROCECURES CELL LINES AND CHEMICALS. HTERT RPE - 1 (HUMAN RETINAL PIGMENTED EPITHELIUM), HCT - 116 (HUMAN COLORECTAL CARCINOMA), HELA S3 (HUMAN CERVICAL CARCINOMA), AND CCD - 112CON (HUMAN COLON) WERE PURCHASED FROM THE AMERICAN TYPE CULTURE COLLECTION (MANASSAS, VA, USA). MCF - 7 (HUMAN BREAST ADENOCARCI NOMA) CELL LINE WAS KINDLY PROVIDED BY DRS. JAVIER BLANCO AND ADOLFO QUIONES LOMBRAA (DEPARTMENT OF PHARMACEUTICAL SCIENCES, UNIVERSITY AT BUFFALO, BUFFALO, NY, USA). CULTURE MEDIA (DMEM, EMEM, AND DMEM/F - 12 50/50), FETAL BOVINE SERUM (FBS), PENICILLIN/S TREPTOMYCIN MIXTURE, AND TRYPSIN WERE BOUGHT FROM CORNING (MANASSAS, VA, USA). DIMETHYLSULFOXIDE (DMSO), 3 - (4,5 - DIMETHYLTHIAZOL - 2 - YL) - 2,5 - DIPHENYLTETRAZOLIUM BROMIDE (MTT ), - TUBULIN ANTIBODY WERE PURCHASED FROM SIGMA - ALDRICH, USA . PARP ANTIBODY WAS PURCHA SED FROM CELL SIGNALING . STAUROSPORINE AND DOXORUBICIN W ERE BOUGHT FROM ENZO AND TOCRIS , AND LIPID STANDARDS FROM AVANTIPOLAR LIPIDS. LC - MS COLUMNS WERE OBTAINED FROM PHENOMENEX. ALL OTHER REAGENTS WERE ACQUIRED FROM SIGMA ALDRICH AND SOLVENTS WERE LC - MS G RADE. CELL CULTURE AND TREATMENTS. HCT - 116, HELA S3 AND MCF - 7 WERE GROWN IN DMEM AND CCD - 112 CELLS IN EMEM. RPE - 1 CELL LINE WAS GROWN IN DMEM/F - 12 50/50 CONTAINING 0.3% ( W /V) SODIUM BICARBONATE. ALL CELL CULTURE MEDIA WERE SUPPLEMENTED WITH 10% (V/V) FBS, 100 U/ML PENICILLIN, AND 100 MG/ML STREPTOMYCIN. CELL LINES WERE MAINTAINED USING STANDARD INCUBATION CONDITIONS AT 37 C AND 5% CO 2 . CELLS WERE TREATED AT 70 - 80% CONFLUENCY. TWENTY - FOUR HOURS AFTER SEEDING, CELLS WERE EXPOSED TO DIFFERENT TIMES AND CONCENTRATIONS OF STAUROSPORINE, DOXORUBICIN, C16 - CERAMIDE OR - DIHYDROCERAMIDE AT 37 C. IMMUNOFLUORESCENCE AND IMAGE ACQUISITION . AFTER 24 HOURS OF SEEDING , CELLS WERE TREATED WITH 0.3 M STA UROSPORINE. AFTER TREATMENT, THE STAINING WAS CARRIED OUT USING THE APOPTOSIS/NECROSIS DETECTION KIT (AB176749, ABCAM, CAMBRIGDE, UK) FOLLOWING MANUFACTURERS INSTRUCTIONS. IMAGES WERE ACQUIRED ON A LEICA DMI6000B INVERTED MICROSCOPE USING LAS AF SOFTWARE (LEICA AF6000 MODULAR SYSTEM, LEICA MICROSYSTEMS CMS GMBH, GERMANY). LC - MS METHOD DATA ANALYSIS. THE FLOW RATE WAS 0.1 ML/MIN FOR THE FIRST 5 MIN FOLLOWED BY A CHANGE TO 0.5 ML/MIN FOR THE REMAINDER OF THE GRADIENT. A DUALJSI FITTED ELECTROSPRAY IONIZATION (ESI) SOURCE WAS USED FOR MS ANALYSIS WITH A CAPILLARY VOLTAGE OF 3500 V AND FRAGMENTOR VOLTAGE OF 175 V. DRYING GAS TEMPERATURE WAS 350 C WITH A FLOW RATE OF 12 L/MIN. DATA WAS COLLECTED USING AN M/Z RANGE OF 50 - 1700 IN EXTENDED DYNAMIC MODE. TANDEM MAS S SPECTROMETRY DATA WERE COLLECTED USING THE FOLLOWING COLLISION ENERGIES: 15, 35, 55, 75, 95 EV FOR EACH M/Z . FOR UNTARGETED LIPIDOMICS, RAW DATA OBTAINED WAS IMPORTED INTO MASSHUNTER PROFINDER (VERSION B.06.00, AGILENT TECHNOLOGIES) FOR PEAK ALIGNMENT. F OR EACH PROFILING EXPERIMENT, FIVE BIOLOGICAL REPLICATES FOR THE THREE CONDITIONS (CONTROL, 5H, AND 16 H) WERE USED . DATA FROM PROFINDER WAS IMPORTED INTO AGILENT MASS PROFILER PROFESSIONAL (MPP, VERSION B12.6.1) FOR STATISTICAL ANALYSIS WHERE SPECIES WERE FILTERED BASED ON FREQUENCY (60%). WE CONDUCTED ANOVA TO DETERMINE STATISTICALLY SIGNIFICANT SPECIES AND ELIMINATED ALL FEATURES WITH P > 0.05. NEXT, SPECIES WERE COMPARED IN A PAIRWISE MANNER USING THREE INDEPENDENT PROFILING EXPERIMENT S AND WE FOCUSED ON THE SPECIES THAT CHANGE D AT LEAST THREE FOLD ACROSS THREE INDEPENDENT PROFILING EXPERIMENTS. THE RESULTING COMPOUNDS WERE THEN MATCHED TO METLIN DATABASE TO IDENTIFY A CANDIDATE MOLECULE BASED ON ACCURATE MASS (TAUTENHAHN ET AL., 2012; ZHU ET AL., 2013) . KNOWN LIPID STANDARDS (OR LIPIDS BELONGING TO SAME LIPID FAMILIES) WERE PURCHASED FOR THE CANDIDATE LIPIDS. MS/MS FRAGMENTATION PATTERNS OF THE SPECIES OF INTEREST AND KNOWN CANDIDATE LIPIDS WERE COMPARED. WHILE FRAGMENTATION PATTERNS WERE INVESTIGATED, SEARCHES BASED ON MS/MS FRAGMENTS PROVIDED IN METLIN WERE USED COMPLEMENTARY TO THE FRAGMENTATION PATTERN OF KNOWN STANDARDS. FOR TARGETED ANALYSIS, THE CORRESPONDING M/Z S FOR EACH ION WERE EXTRACT ED IN AGILENT MASSHUNTER QUALITATIVE ANALYSIS (VERSION B.06.00). PEAK AREAS FOR EACH ION WERE MANUALLY INTEGRATED AND AVERAGE ABUNDANCES WERE CALCULATED FOR EACH CONDITION. FOLD CHANGES WERE SUBSEQUENTLY CALCULATED. DROPLET DIGITAL PCR (DDPCR). QUANTIFICAT ION WAS PERFORMED WITH PROBE - BASED ASSAYS USING PRE - DESIGNED INTEGRATED DNA TECHNOLOGIES (IDT, CORALVILLE, IA, USA) PRIMERS AND ZEN DOUBLE - QUENCHED PROBES. WATER - IN - OIL EMULSION DROPLETS WERE GENERATED USING AN AUTOMATED DROPLET GENERATOR (BIO - RAD) AND TRA NSFERRED TO 96 - WELL PLATES, WHICH WERE HEAT - SEALED USING FOIL SHEETS. TARGET GENES AND THE REFERENCE GENE, HPRT1 (HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE 1) WERE AMPLIFIED IN PARALLEL BY THERMAL CYCLING THE DROPLET EMULSIONS AS FOLLOWS: 95 C FOR 10 MIN ( TAQ DNA POLYMERASE ACTIVATION), 40 CYCLES OF 94 C FOR 30 S (DENATURATION), 56 C FOR 60 S (ANNEALING AND EXTENSION) WITH A FINAL 10 MIN INACTIVATION STEP AT 98 C. THE FLUORESCENCE OF EACH THERMAL LY CYCLED DROPLET WAS MEASURED USING THE QX200 DROPLET READ ER (BIO - RAD). DATA WAS ANALYZED USING THE QUANTASOFT SOFTWARE (BIO - RAD) AFTER THRESHOLD SETTING ON FLUORESCENCE OF NEGATIVE CONTROLS. SEQUENCES OF PRIMERS: CERS1 SENSE 5 - GCC TTC CAC AAC CTC CTG - 3, ANTISENSE 5 - AAC TGG GTA ACA AGC AGA GTC - 3; CERS2 SENSE 5 - CA C TGC GTT CAT S 11 CTT CTA CCA - 3, ANTISENSE 5 - GCT CTA TCC TGC CTT CTT TGG - 3; CERS3 SENSE 5 - ACA TCA AAG CCA AGT CTA AAT AAC AG - 3, ANTISENSE 5 - GGC TAT ATG ACT TAT GGG AGG TT - 3; CERS4 SENSE 5 - ACA TCA GAA GCC CGT TGA AG - 3, ANTISENSE 5 - CTC TTC CTC ATC TTC TCC TTT GT C - 3; CERS5 SENSE 5 - CCG ATT ATC TCC CAA CTC TCA A - 3, ANTISENSE 5 - GCC AAT TAT GCC AAG TAT CAG C - 3; CERS6 SENSE 5 - TGA CTC CGT AGG TAA ATA CAT AAA GG - 3, ANTISENSE 5 - CAA TCA GGA GAA GCC AAG CA - 3; HPRT1 SENSE 5 - TTG TTG TAG GAT ATG CCC TTG A - 3, ANTISENSE 5 - GCG AT G TCA ATA GGA CTC CAG - 3. MTT ASSAY. AFTER TREATMENT, THE PLATES WERE CENTRIFUGED, THE MEDIUM WAS REMOVED AND 200 L OF MEDIA WITH 9% MTT (5 MG/ML IN PBS) WERE ADDED TO EACH WELL. THE PLATES WERE SUBSEQUENTLY INCUBATED FOR 3 H AT 37 C. AFTER INCUBATION, 15 0 L OF MEDIA WERE REMOVED FROM EACH WELL AND 90 L OF DMSO WERE ADDED TO EACH WELL. TREATMENTS WERE CARRIED OUT AT A MINIMUM OF TRIPLICATES. THE ABSORBANCE WAS MEASURED USING AN AUTOMATIC PLATE READER (SPECTRA MR, DYNEX TECHNOLOGIES, INC., CHANTILLY, VA, USA) AT 550 NM. CELL VIABILITY WAS CALCULATED AS PERCENTAGES RELATIVE TO CONTROL CELLS. WESTERN BLOT ANALYSIS. CELLS AND MEDIA WERE COLLECTED IN FALCON TUBES AND CENTRIFUGED. THE SUPERNATANT WAS DISCARDED AND CELLS WERE WASHED WITH PBS, AND CENTRIFUGED. C ELLS WERE LYSED WITH M - PER REAGENT (THERMO SCIENTIFIC, ROCKFORD, IL, USA) CONTAINING A PROTEASE INHIBITOR COCKTAIL (ROCHE DIAGNOSTICS INDIANAPOLIS, IN, USA); DEBRIS WAS THEN REMOVED BY CENTRIFUGATION AND SUPERNATANT USED FOR MEASURING PROTEIN CONCENTRATION BY BRADFORD ASSAY (THERMO SCIENTIFIC, ROCKFORD, IL, USA). EQUIVALENT AMOUNTS OF PROTEIN WERE SEPARATED ON 10% SDS - PAGE AND TRANSFERRED ONTO A NITROCELLULOSE MEMBRANE (BIO - RAD LABORATORIES, GERMANY). MEMBRANES WERE BLOCKED WITH TBS - TWEEN [10 MM TRIS - BASE, 100 MM NACL, 0.1% TWEEN 20 (PH 7.5)] CONTAINING 5% NONFAT DRY MILK, THEN WASHED WITH TBS - TWEEN AND EXPOSED TO ANTI - PARP (PARP RABBIT AB, CELL SIGNALING TECHNOLOGY, DANVERS, MA, USA). AFTER PRIMARY INCUBATION, MEMBRANES WERE WASHED WITH TBS - TWEEN FOLLOWED B Y INCUBATION WITH ANTI - RABBIT SECONDARY ANTIBODY. THEN, MEMBRANES WERE WASHED WITH TBS - TWEEN, AND DEVELOPED USING THE SUPERSIGNAL WEST PICO KIT (THERMO SCIENTIFIC, ROCKFORD, IL, USA). BLOTS WERE STRIPPED USING STRIPPING BUFFER [6.3 MMOL/LTRIS - BASE, 0.2% SD S, 0.8 % (V/V) - MERCAPTOETHANO L (PH 6.8)] AND RE - BLOTTED FOR ANTI - TUBULIN IN ORDER TO CONFIRM EQUAL PROTEIN LOADING. NORMALIZATION AND THE MATRIX EFFECT. N ORMALIZATION ACROSS DIFFERENT SAMPLES WAS CARRIED OUT BASED ON PROTEIN CONCENTRATION AND THE USE OF INTERNAL STANDARDS (OLEIC ACID - 13 C18 AND C17 - CERAMIDE). IONIZATION EFFICIENCIES BASED ON TOTAL ION COUNTS OF THE STANDARDS WERE INVESTIGATED AND CORRECTED WHEN NECESSARY. QUANTIFICATION OF CERAMIDES BY LC - MS. A DILUTION EXPERIMENT WAS PERFORMED WITH A NON - ENDOGENOUS CERAMIDE TO QUANTIFY CERAMIDES IN HCT - 116 AND CCD - 112 CELLS. PRIOR TO EXTRACTION, SAMPLES WERE SPIKED WITH C17 - CERAMIDE AS AN INTERNAL STANDARD. AFTER EXTRACTION, SERIAL DILUTIONS OF THE SAMPLES WERE PERFORMED. THESE SAMPLES WERE ANALYZED USING LC - MS QTOF IN NEGATIVE ESI MODE. THE DATA ACQUISITION CONDITIONS WERE SIMILAR TO THAT OF PROFILING EXPERIMENTS. A CALIBRATION CURVE WAS OBTAINED BASED ON THE ION COUNTS AND DIFFERENT CONCENTRATIONS OF C17 - CERAMIDE IN HCT - 116 AND CCD - 112 CELLS SEPARATELY. IN ORDER TO USE C17 - CERAMIDE AS A STANDARD TO CALCULATE THE CONCENTRATIONS OF ENDOGENOUS CERAMIDES, EQUAL CONCENTRATIONS OF C14 - , C17 - , C16 - , C22 - AND C24 - CERAMIDE WERE PREPARED, INJECTED INTO LC - MS QTOF AND ION ABUNDANCES WERE CALCULATED. BASED ON THIS , R ESPONSE FACTORS THAT ACCOUNT FOR DIFFERENCES IN IONIZATION EFFICIENCY WERE DETERMINED FOR C14 - , C16 - , C22 - AND C24 - CERAMIDES. SUBSEQUENTLY, C14 - , C16 - , C22 - AND C24 - CERAMIDE WERE QUANTIFIED. MEDIA EXTRACTION. THE GROWTH MEDIA OF STAUROSPORINE - TREATED AND UNTREATED HCT - 116 WERE COLLECTED FROM WHICH LIPIDS WERE EXTRACTED. BRIEFLY, A MIXTURE OF MEDIA:MEOH:CHCL 3 (1:1:2) WAS VORTEXED AND ALLOWED TO SETTLE (THE PROCEDURE WAS REPEATED THREE TIMES PER SAMPLE). THE SAMPLE WAS THEN CENTRIFUGED AND THE ORGANIC LAYER RECOVERED FROM WHICH 6 ML WERE TAKEN AND DRIED UNDER VACUUM. FINALLY, ALL THE SAMPLES WERE RESUSPENDED IN 200 L OF CHLOROFORM. THE EXPERIMENT WAS CARRIED OUT IN TRIPLICATE. S 12 SUPPLEMENTAL LIPID ASSIGNMENTS R T CORRESPONDS TO RETENTION TIME . STANDARDS. C14 - CERAMIDE . FRAGMENT S GENERATED IN NEGATIVE IONIZATION MODE. [M - H] - M/Z : OBSERVED 508.4797; THEORETICAL 508.4735. OBSERVED FRAGMENTS: 478.4704, 476.4534, 460.4570, 268.2360, 263.2456, 252.2416, 237.2315, 226.2232, 209.1996. FRAGMENTS GENERATED IN POSITIVE IONIZATION MODE . [M+H - H 2 O] + M/Z : OBSERVED 492.4790; THEORETICAL 492.4775. OBSERVED FRAGMENTS: 474.4668, 462.4668, 282.2791, 264.2689, 228.2319. C14 - DIHYDROCERAMIDE . FRAGMENTS GENERATED IN NEGATIVE IONIZATION MODE. [M - H] - M/Z : OBSERVED 510.4982; THEORETICAL 510.4892. OBSERVED FRAGMENTS: 478.4734, 462.4790, 268.2393, 265.2601, 252.2445, 239.2481, 226.2295, 209.2024. FRAGMENTS GENERATED IN POSITIVE IONIZATION MODE. [M+H - H 2 O] + M/Z : OBSERVED 494.4936; THEORETICAL 494.4932. OBSERVED FRAGMENTS: 476.4817, 464.4785, 284.2946, 266.2833, 228.2327. C22 - CERAMIDE . FRAGMENTS GENERATED IN NEGATIVE IONIZATION MODE. [M - H] - M/Z : OBSERVED 620.6047; THEORETICAL 620.5987. OBSERVED FRAGMENTS: 590.595 3, 588.5782, 572.5801, 380.3618, 364.3666, 338.3483, 321.3252, 263.2442, 237.2310. FRAGMENTS GENERATED IN POSITIVE IONIZATION MODE. [M+H - H 2 O] + M/Z : OBSERVED 604.6036; THEORETICAL 604.6027. OBSERVED FRAGMENTS: 586.5912, 574.5915, 340.3574, 282.2792, 264.26 93. C16 - G LUCOSYLCERAMIDE . FRAGMENTS GENERATED IN NEGATIVE IONIZATION MODE. [M - H] - M/Z : OBSERVED 698.5570; THEORETICAL 698.5576. OBSERVED FRAGMENTS: 536.5096, 280.2760, 263.2442, 255.2494, 237.2379, 179.0699. FRAGMENTS GENERATED IN POSITIVE IONIZATION MODE. [M+H - H 2 O] + M/Z : OBSERVED 682.5622; THEORETICAL 682.5616. OBSERVED FRAGMENTS: 664.5094, 520.5079, 502.4980, 282.2783, 264.2685, 256.2636, 145.0501, 121.0999, 95.0855. IDENTIFICATION OF SPECIES FROM GLOBAL UN TARGETED PROFILING IDENTIFICATION OF M/Z : 360.3093 (C18 FA DERIVATIVE) . RT 47.4 . MS/MS FRAGMENTS OBTAINED WERE SEARCHED IN METLIN. [M+H+H 2 O] + M/Z : OBSERVED 360.3093; THEORETICAL 360.3108. OBSERVED FRAGMENTS: 342.3014, 284.2903, 267.2781, 266.2920, 76.0398. IDENTIFICATION OF M/Z : 398.3278 (C16:1 ACYLCARNITINE ). RT 40.3 . MS/MS FRAGMENTS OBTAINED WERE SEARCHED IN METLIN. [M] + M/Z : OBSERVED 492.4778; THEORETICAL 492.4775. OBSERVED FRAGMENTS: 339.2516, 255.2351, 237.2206, 144.1036, 85.0332, 60.0848. IDENTIFICATION OF M/Z : 492.4772 ( C14 - CERAMIDE). RT 56.6 . FRAGMENTATION PATTERN WA S COMPARED WITH CERAMIDE STANDARDS. [M+H - H 2 O] + M/Z : OBSERVED 492.4778; THEORETICAL 492.4775. OBSERVED FRAGMENTS: 474.4666, 462.4638, 282.2787, 264.2683, 228.2313. IDENTIFICATION OF M/Z : 510.4841 (C14 - DIHYDROCERAMIDE) . RT 61.5. FRAGMENTATION PATTERN WA S COMPARED WITH DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 510.4810; THEORETICAL 510.4892. OBSERVED FRAGMENTS: 478.4758, 462.4535, 268.2292, 252.2382, 239.2356, 226.2160, 209.1900. IDENTIFICATION OF M/Z: 536.5007 (C16 - CERAMIDE) . RT 62.7. FRAGMENTATION PATTERN WAS COMPARED WITH CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 536.5042; THEORETICAL 536.5048. OBSERVED FRAGMENTS: 506.4955, 504.4794, 488.4835, 280.2646, 237.2227. IDENTIFICATION OF M/Z: 538.5163 (C16 - DIHYDROCERAMIDE) . RT 63.2. FRAGMENTATION PATTERN WAS COMPARED WITH DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 538.5188; THEORETICAL 538.5205. OBSERVED FRAGMENTS: 506.4920, 490.4976, 296.2584, 280.2637, 265.2537, 254.2487, 239.2375, 237.2213. IDENTIFICATION OF M/Z: 570.4603 (C16:1 - CERAMIDE) . RT 61.2. FRAGMENTATION PATTERN WAS COMPARED WITH CERAMIDE STANDARDS. [M - CL] - M/Z : OBSERVED 570.4691; THEORETICAL 570.4658. OBSERVED FRAGMENTS: 237.2270, 235.2088. S 13 IDENTIFICATION OF M/Z: 624.6283 (C22 - DIHYDROCERAMIDE) . RT 61.2. FRAGMENTATION PATTERN WAS COMPARED WITH DIHYDROCERAMIDE STANDARD. [M+H] + M/Z : OBSERVED 624.6281; THEORETICAL 624.6289. OBSERVED FRAGMENTS: 606.6179, 588.6074, 340.3556, 302.3051, 284.2955, 266.2855. IDENTIFICATION OF M/Z: 630.6178 (C24:1 - CERAMIDE) . RT 61.7. FRAGMENTATION PATTERN WAS COMPARED WITH CERAMIDE STANDARDS. [M+H - H 2 O] + M/Z : OBSERVED 630.6194; THEORETICAL 630.6184. OBSERVED FRAGMENTS: 612.6081, 600.6074, 366.3728, 282.2787, 264.2687. IDENTIFICATION OF M/Z: 648.6231 (C24:1 - DIDHYDROCERAMIDE) . RT 68.2. FRAGMENTATION PATTERN WAS COMPARED WITH DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 648.6250; THEORETICAL 648.6300. OBSERVED FRAGMENTS: 616.5997, 600.6116, 406.3619, 390.3762, 364.3541, 347.3308, 265.2378, 239.2403. THE COMPOUND WAS IDENTIFIED AS CER(18:0/24:1). IDENTIFICATI ON OF M/Z: 658.6477 (C26:1 - CERAMIDE) . RT 62.6. FRAGMENTATION PATTERN WAS COMPARED WITH CERAMIDE STANDARDS. [M+H - H 2 O] + M/Z : OBSERVED 658.6497; THEORETICAL 658.6497. OBSERVED FRAGMENTS: 640.6387, 628.6442, 340.3574, 394.4079, 282.2813, 264.2708 . THE COMPOUND WAS IDENTIFIED AS CER(18:1/26:1). IDENTIFICATION OF M/Z: 677.5607 (C14 - DIHYDROSPHINGOMYELIN) . RT 54.9. FRAGMENTATION PATTERN WAS COMPARED WITH DIHYDROCERAMIDE STANDARD AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 677.5609; THEORETICAL 677.5592. OBSERVED FRAGMENTS: 659.5458, 600.4703, 494.4903, 252.2352, 266.2886, 228.2402, 184.0779, 166.0669, 125.0038, 86.1004. IDENTIFICATION OF M/Z: 810.6744 (C24:1 - HEXOSYLDIHYDROCERAMIDE) . RT 66.4. FRAGMENTATION PATTER N WAS COMPARED WITH HEXOSE - AND DIHYDRO - CERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 810.6802; THEORETICAL 810.6828. OBSERVED FRAGMENTS: 752.5916, 648.6291, 616.6018, 600.6096, 406.3673, 390.3807, 364.3539, 347.3375, 265.2550, 239.2319, 179.0616, 161.0478. IDENTIFICATION OF M/Z: 815.7018 (C24:1 - DIHYDROSPHINGOMYELIN) . RT 60.8. FRAGMENTATION PATTERN WAS COMPARED WITH DIHYDROCERAMIDE STANDARD AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 815.7020; THEORETICAL 815.7001. OBSERVED FRAGMENTS: 797.6849, 738.6064, 632.6289, 614.6190, 284.2913, 266.2842, 184.0740, 166.0628, 125.0000, 86.0971. IDENTIFICATION OF M/Z: 817.7147 (C24 - DIHYDROSPHINGOMYELIN) . RT 61.4. FRAGMENTATION PATTERN WAS COMPARED WITH DIHYDROCERAMIDE STANDARD AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 817.7120; THEORETICAL 817.7157. OBSERVED FRAGMENTS: 799.7124, 740.6381, 676.6314, 634.6362, 368.3945, 266.2840, 184.0730, 166.0648, 125.0005, 86.0990. IDENTIFICATION OF M/Z: 841.7160 (C26:1 - SPHINGOMYELIN) . RT 61.2. FRAGMENTATION PATTERN WAS COMPARED WITH CERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 841.7130; THEORETICAL 841.7157. OBSERVED FRAGMENTS: 82 3.7032, 764.6304, 658.6466, 640.6365, 418.3986, 264.2655, 184.0710, 166.0573, 124.9972, 86.0947. IDENTIFICATION OF SPECIES FROM TARGETED ANALYSIS RT CORRESPONDS TO RETENTION TIMES. CERAMIDES C14 - C ERAMIDE . RT 60.9. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 508.4698; THEORETICAL 508.4735. OBSERVED FRAGMENTS: 476.4566, 460.4527, 252.2295, 237.2184, 226.2047, 209.1992. C 16 - C ERAMIDE . RT 62.7. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 536.5044; THEORETICAL 536.5048. OBSERVED FRAGMENTS: 506.4897, 504.4753, 488.4793, 280.2627, 237.2202. C18 - C ERAMIDE . RT 64.3. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION P ATTERN FOUND BY THE CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 564.5319; THEORETICAL 564.5361. OBSERVED FRAGMENTS: 534.5205, 532.5055, 516.5113, 308.2927, 282.2789, 265.2523, 237.2217. S 14 C20 - C ERAMIDE . RT 65.7. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 592.5640; THEORETICAL 592.5674. OBSERVED FRAGMENTS: 562.5579, 560.5370, 544.5417, 336.3249, 310.3076, 293.2841, 237.2233. C22 - C ERAMIDE . RT 66.2. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 620.5944; THEORETICAL 620.5987. OBSERVED FRAGMENTS: 590.5822, 588.5648, 572.5729, 364.3553, 338.3398, 321.3110, 237.2211. C24 - C ERAMIDE . RT 67.3. THE COMPOUND WAS ASSIGNED BAS ED ON THE FRAGMENTATION PATTERN FOUND BY THE CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 648.6245; THEORETICAL 648.6300. OBSERVED FRAGMENTS: 618.6138, 616.5990, 600.6056, 392.3960, 237.2203. C26 - C ERAMIDE . RT 69.1. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGM ENTATION PATTERN FOUND BY THE CERAMIDE STANDARDS. [M - H] - M/Z : OBSERVED 676.6655; THEORETICAL 676.6613. OBSERVED FRAGMENTS: 646.6543, 644.6414, 628.6500, 420.4269, 394.4041, 377.3809, 237.2313. DIHYDROCERAMIDES C14 - D IHYDROCERAMIDE. RT 61.5. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 510.4918; THEORETICAL 510.4892. OBSERVED FRAGMENTS: 478.4624, 462.4692, 252.2374, 239.2470, 209.1973. C16 - DIHYDROCERAMIDE. RT 6 3.2 . T HE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 538.5211; THEORETICAL 538.5205. OBSERVED FRAGMENTS: 506.4965, 490.5012, 280.2676, 239.2412, 237.2254. C18 - DIHYDROCERAMIDE. RT 64.7. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 566.5568; THEORETICAL 566.5518. OBSERVED FRAGMENTS: 534.5300, 518.5332, 308.3019, 265.2575, 239.2459. C20 - DIHYDROCERAMIDE. RT 66.1. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 594.5879; THEORETICAL 594.5831. OBSERVED FRAGMENTS: 564.5659, 562.5659, 336.3357, 293.2909, 239.2459. C22 - DIHYDROCERAMIDE. RT 67.3. THE CO MPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE DIHYDROCERAMIDE STANDARD. [M - H] - M/Z: OBSERVED 622.6212; THEORETICAL 622.6144. OBSERVED FRAGMENTS: 590.5906, 574.5965, 364.3657, 321.3210, 239.2448. C24 - DIHYDROCERAMIDE. RT 68.5. THE COMPO UND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY THE DIHYDROCERAMIDE STANDARD. [M - H] - M/Z : OBSERVED 650.6480; THEORETICAL 650.6457. OBSERVED FRAGMENTS: 618.6222, 602.6281, 392.3967, 349.3528, 239.2403. SPHINGOMYELINS C14 - SPHINGOMYELIN. RT 53.8. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY CERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 675.5419; THEORETICAL 675.5436. OBSERVED FRAGMENTS: 657.5286, 598.4701, 474.4646, 264.2 683, 252.2326, 184.0725, 166.0622, 124.9995, 86.0967. C16 - SPHINGOMYELIN. RT 55.3. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY CERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 703.57 22; THEORETICAL 703.5749. OBSERVED FRAGMENTS: 685.5635, 626.4875, 502.4933, 280.2684, 264.2688, 184.0740, 166.0635, 125.0005, 86.0981. C18 - SPHINGOMYELIN. RT 56.8. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY CERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 731.6091; THEORETICAL 731.6062. OBSERVED FRAGMENTS: 713.6046, 530.5130, 264.2700, 184.0736, 166.0620, 124.9999, 86.0973. C22 - SPHINGOMYELIN. RT 59.5. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY CERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 787.6664; THEORETICAL 787.6688. OBSERVED FRAGMENTS: 769.6508, 603.5366, 364.3449, 264.2676, 184.0739, 166.0603, 125.0003 , 86.0980. S 15 C24 - SPHINGOMYELIN. RT 61.1. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY CERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 815.7054; THEORETICAL 815.7001. OBSERVED FRAGMENT S: 797.6888, 738.6164, 674.6172, 632.6277, 614.6227, 392.3890, 264.2705, 184.0743, 166.0620, 125.0004, 86.0974 . C24 - SPHINGOMYELIN. RT 65.1. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY CERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDE D IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 843.7285; THEORETICAL 843.7314. OBSERVED FRAGMENTS: 825.6981, 642.6367, 264.2674, 184.0695, 166.0595, 124.9967, 86.0944. DIHYDROSPHINGOMYELINS C14 - DIHYDROSPHINGOMYELIN. RT 54.9. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY DIHYDROCERAMIDE STANDARD AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 677.5522; THEORETICAL 677.5592. OBSERVED FRAGMENTS: 659.4223, 600.4842, 494.4885, 266.2783, 252.2352, 184.0718, 166.0602, 124.9968, 86.0954. C16 - DIHYDROSPHINGOMYELIN. RT 56.5. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATTERN FOUND BY DIHYDROCERAMIDE STANDARD AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M /Z : OBSERVED 705.5893; THEORETICAL 705.5905. OBSERVED FRAGMENTS: 687.5880, 628.5065, 522.5313, 504.5008, 280.2644, 266.2814, 184.0733, 166.0619, 124.9999, 86.0973. C24 - DIHYDROSPHINGOMYELIN. RT 61.6. THE COMPOUND WAS ASSIGNED BASED ON THE FRAGMENTATION PATT ERN FOUND BY DIHYDROCERAMIDE STANDARDS AND MS/MS FRAGMENTS PROVIDED IN METLIN METABOLITE DATABASE. [M] + M/Z : OBSERVED 817.7120; THEORETICAL 817.7157. OBSERVED FRAGMENTS: 799.7124, 740.6381, 676.6314, 634.6362, 368.3945, 266.2840, 184.0730, 166.0648, 125.00 05, 86.0990. S 16 SUPPLEMENTAL REFERENCES TAUTENHAHN, R., CHO, K., URITBOONTHAI, W., ZHU, Z., PATTI, G.J., AND SIUZDAK, G. (2012). AN ACCELERATED WORKFLOW FOR UNTARGETED METABOLOMICS USING THE METLIN DATABASE. NAT BIOTECHNOL 30 , 826 - 828. ZHU, Z.J., SCHULTZ, A.W., WANG, J., JOHNSON, C.H., YANNONE, S.M., PATTI, G.J., AND SIUZDAK, G. (2013). LIQUID CHROMATOGRAPHY QUADRUPOLE TIME - OF - FLIGHT MASS SPECTROMETRY CHARACTERIZATION OF METABOLITES GUIDED BY THE MET LIN DATABASE. NAT PROTOC 8 , 451 - 460.