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Supplemental Information Membrane Disruption, But Supplemental Information Grunt et al. SUPPLEMENTAL INFORMATION MEMBRANE DISRUPTION, BUT NOT METABOLIC REWIRING, IS THE KEY MECHANISM OF ANTICANCER-ACTION OF FASN-INHIBITORS – A MULTI- OMICS ANALYSIS IN OVARIAN CANCER Thomas W. Grunt1,2,3,*, Astrid Slany4, Mariya Semkova4, Ramón Colomer5, María Luz López- Rodríguez6, Michael Wuczkowski7, Renate Wagner1,2, Christopher Gerner4, Gerald Stübiger2,7 1Cell Signaling & Metabolism Networks Program, Division of Oncology, Department of Medicine I, Medical University of Vienna, Austria; 2Comprehensive Cancer Center, Vienna, Austria; 3Ludwig Boltzmann Institute for Hematology & Oncology, Vienna, Austria; 4Department of Analytical Chemistry, University of Vienna, Austria; 5Department of Medical Oncology, Hospital Universitario La Princesa and Spanish National Cancer Research Centre (CNIO), Clinical Research Program, Madrid, Spain; 6Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Spain; 7Department of Biomedical Imaging & Image-guided Therapy, Medical University of Vienna, Austria *Corresponding author: Thomas W. Grunt, Division of Oncology, Department of Medicine I, Medical University of Vienna, Waehringer Guertel 18 – 20, A-1090 Vienna, Austria; P: +43 (0)1 40400-54570, F: +43 (0)1 40400-54650, Email: [email protected] 1 Supplemental Information Grunt et al. SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1. Influence of FASN-inhibition on total amount of lipids per cell in (a) SKOV3 and (b) OVCAR3 cells, and (c) scheme of the action of FASN in de novo lipogenesis and the synthesis of the major cellular lipid classes. Relative change of total cellular lipids in (a) SKOV3 and (b) OVCAR3 cells upon G28UCM treatment (20µM, 72 hours). A decrease of 60% (SKOV3) to >80% (OVCAR3) in cell number and of about 30% lipid content was observed compared to cells grown in 0.1% DMSO. (c) Palmitate (16:0) as the primary reaction product of FASN is further processed to acyl-CoA via several steps of elongation and desaturation leading to fatty acids with different carbon chain-lengths and number of double bonds. These fatty acids are then esterified to cholesterol or glycerol to generate cholesterol ester (CE), triacylglycerol (TAG), diacylglycerol (DAG) and phospholipids (PL) as the major structural (e.g. membrane), signalling and storage lipid classes. Supplemental Figure S2. Representative MALDI mass spectra of lipid extracts of untreated and G28UCM treated SKOV3 (a, b) and OVCAR3 (c, d) recorded in positive (a, c) and negative (b, d) ionization mode at 8 hours and 24 hours. Phospholipid class specific internal standards (indicated by asterisks) were added to the samples for semiquantitative analysis of the corresponding lipid species. Abbreviations: LPC, lysophosphatidylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidyglycerol; PI, phosphatidylinositol; PS, phosphatidylserine Supplemental Figure S3. Reproducibility of glycerophospholipid analysis by MALDI-MS. Six individual control cell culture samples were prepared according to the lipid extraction and MS analysis protocols described in Material and Methods. In (a) the data from the independent analysis of the lipid extracts displaying the relative abundances of the sum of individual lipid species relative to class specific internal standards (Ratio vs. Int. Std.) are displayed and in (b) their means ± SD are shown. Numbers above the error bars indicate the coefficient of variation (CV %). Abbreviations: CL, cardiolipin; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin. Supplemental Figure S4. Effects of FASN-inhibition on the phosphatidylcholine (PC) composition of SKOV3 and OVCAR3 cells. Changes in the relative composition of PC species containing fatty acid residues with 0-2 total double bonds (DBs) in (a) SKOV3 and (b) OVCAR3 cells treated with 0.1% DMSO and 40µM G28UCM for 8 hours and 24 hours. Displayed is the relative composition of PC 2 Supplemental Information Grunt et al. species with 0-2 DBs in % of total PC. Values are means ± SD (n = 3). Letter code of the fatty acid residues: L, linoleate (18:2); O, oleate (18:1); P, palmitate (16:0); S, stearate (18:0). SUPPLEMENTAL TABLE LEGENDS Supplemental Table S1: Reproducibility analysis of biological and technical replicates of SKOV3 samples. Glycerophospholipids were quantitatively measured by LC-ESI-MS/MS according to the AbsoluteIDQ p180 kit (see Material and Methods). Samples of three independent cell cultures (biological replica) treated with 0.1% DMSO (control) or 40µM G28UCM for 8 hours and 24 hours were measured two times each (technical replica). From that the standard deviation (SD) and coefficient of variation (CV) for the individual lipid species were calculated. Supplemental Table S2: Shotgun proteomic analysis of SKOV3 (a) and OVCAR3 (b) cells exposed for 8 hours and 24 hours to 40µM G28UCM. All proteins identified by MS/MS. Colour Code: 8h Up 24h Up 8h Down 24h Down Supplemental Table S3: DAVID-assisted shotgun proteomic analysis of key cell processes in SKOV3 and OVCAR3 cells exposed to 40µM G28UCM using BioCarta and KEGG databases. Legend Significantly (p < 0.05) downregulated proteins: Blue cell Significantly (p < 0.05) upregulated proteins: Red cell For Individual Proteins SKOV3/OVCAR3 Matching Score: 1,00: uniform regulation at the same time in both cell lines. 0,00: no uniform regulation in both cell lines at the specific time. For Key Cell Processes or Sub-Processes Mean SKOV3/OVCAR3 Matching Score: Mean of 'Individual Protein SKOV3/OVCAR3 Matching Scores' Supplemental Table S4: Summary of all down- (< 50 % of Control) and up-regulated (> 150 % of Control) phosphoproteins as determined by antibody microarray kinomic analysis in SKOV3 (a) and OVCAR3 (b) cells exposed for 24 hours to 40μM G28UCM. 3 Supplemental Figure S1 100 100 Total Amount of Lipids/Cell a 90 b 90 80 Total Number of Cells 80 70 70 60 60 Control Control 50 50 40 40 % of % % of % 30 30 20 20 10 10 0 0 Control G28UCM Conrtrol G28UCM SKOV3 OVCAR3 c Phospholipids (PL) FASN Acylglycerols (TAG, DAG) Cholesterol Ester (CE) ATP+CoA AMP+PPi Palmitate Palmitoyl-CoA Esterification 16:0 Acyl-CoA-Synthetase Elongation Desaturation Acyl-CoA (e.g. Oleate 18:1) Supplemental Figure S2 a c %Int. PC %Int. 678.5* 760.6 510.4* LPC PC 100 LPC 100 * 678.5 760.6 G28UCM 24h 663.4 G28UCM 24h 50 510.4* 761.6 50 732.5 496.3 664.4 782.7 786.6 0 810.7 0 810.6 100 100 * 50 Control 24h 50 Control 24h * 0 * 0 100 100 * 50 * G28UCM 8h 50 G28UCM 8h * 0 0 100 100 * 50 * Control 8h 50 Control 8h 0 0 500 550 600 650 700 750 800 850 900 950 500 550 600 650 700 750 800 850 900 950 m/z m/z %Int. b %Int. PS 687.6 744.6 PI d PG 728.5 PI 100 PS 100 PE PE 701.6 * G28UCM 24h 678.4 G28UCM 24h 647.3 745.6 885.6 793.5* 50 793.6* 50 665.4* 591.5 634.4* 856.5 634.5* 678.4* 729.6 886.6 591.4 766.5 887.6 835.6 913.5 716.5 0 0 911.6 100 100 * * * Control 24h 50 Control 24h 50 * * 0 * * 0 100 100 * * 50 G28UCM 8h 50 * G28UCM 8h * * 0 * 0 100 100 * * * 50 * * Control 8h 50 Control 8h * * 0 0 550 600 650 700 750 800 850 900 950 1000 550 600 650 700 750 800 850 900 950 1000 m/z m/z Supplemental Figure S3 a 3.00 2.50 . Extract #1 2.00 Extract #2 Int.Std 1.50 Extract #3 Extract #4 1.00 Extract #5 Ratio vs. Extract #6 0.50 0.00 LPC SM PC PE PS PG PI CL b 2.50 25.0 2.00 . 19.3 1.50 Int.Std 25.6 1.00 Ratio vs. 12.0 0.50 10.5 28.9 33.3 21.4 0.00 LPC SM PC PE PS PG PI CL Supplemental Figure S4 a 70.0 60.0 60.0 Control 8h SKOV3 Control 24h SKOV3 50.0 G28UCM 8h 50.0 G28UCM 24h PCs PCs 40.0 40.0 30.0 Total Total 30.0 of of 20.0 % 20.0 % 10.0 10.0 0.0 0.0 30.0 35.0 b Control 8h 25.0 OVCAR3 30.0 Control 24h OVCAR3 G28UCM 8h 25.0 G28UCM 24h 20.0 PCs PCs 20.0 15.0 Total Total 15.0 of 10.0 of 10.0 % % 5.0 5.0 0.0 0.0 Supplemental Table S1 Supplemental Table S1: Reproducibility analysis of biological and technical replicates of SKOV3 samples 8h samples 1 - SKOV con1 8h 2 - SKOV con2 8h 3 - SKOV con3 8h 4 - SKOV FASN1 8h 5 - SKOV FASN2 8h control control control G28UCM G28UCM lysoPC a C14:0 8,07 9,2 7,75 8,44 11 lysoPC a C16:0 8,33 6,65 9,39 6,52 10,3 lysoPC a C16:1 14,6 19 17,7 15,8 23,6 lysoPC a C17:0 0,622 0,569 0,678 0,503 0,62 lysoPC a C18:0 1,48 0,836 1,71 0,993 1,59 lysoPC a C18:1 50,1 39,3 49,7 43,3 61,6 lysoPC a C18:2 4,7 5,25 5,01 4,45 7,22 lysoPC a C20:3 1,99 1,64 1,97 1,74 2,69 lysoPC a C20:4 4,42 4,87 5,25 3,56 5,07 lysoPC a C24:0 0,852 0,904 1,37 0,689 0,963 lysoPC a C26:0 1,69 1,56 2,83 1,65 2,27 lysoPC a C26:1 0,422 0,373 0,656 0,351 0,418 lysoPC a C28:0 2,22 1,23 2,53 1,31 2,02 lysoPC a C28:1 0,988 0,812 1,37 0,848 1,15 PC aa C24:0 0,269 0,483 0,459 0,309 0,476 PC aa C26:0 1,94 2,36 2,83 2,2 3,24 PC aa C28:1 1,18 1,47 1,69 1,1 1,73 PC aa C30:0 11,9 9,38 13,2 10,9 12,1 PC aa C30:2 0,147 0,164 0,35 0,16 0,359 PC aa C32:0 29,8 16,4 37 23,4 19,4 PC aa C32:1 79,2 45,6 97,5 58,3 88,2 PC aa C32:2 5,71 3,41 7,74 3,82 5,89 PC aa C32:3 0,339 0,296 0,558 0,235 0,362 PC aa C34:1 285 163 343 224 305 PC aa C34:2 47,6 28,9 61,8 34,5 54,6 PC aa C34:3 3,41 2,01 4,39 2,31 3,67 PC aa C34:4 0,452 0,281 0,511 0,274 0,406 PC aa C36:0 2,51 1,57 3,16 2,02 2,69 PC aa C36:1 33,6 16,8 33,9 30,1 28 PC aa C36:2 75,8 46,9 102 65 93,5 PC aa C36:3 14,9 8,83 18,4 11,7 18,5 1 Supplemental Table S1 PC aa C36:4 9,44 5,08 10,9 5,89 9,37 PC aa C36:5 3,36 1,99 4,02 2,27 3,67 PC aa C36:6 0,524 0,37 0,569 0,371 0,494 PC aa C38:0 3 2,1 4,2 2,51 3,73 PC aa C38:1 1,39 0,71 1,39 1,4
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