Figure File Name: 10.1007_s00018-019-03227-w_Fig5 Technique/Assay: Number 10 = Seahorse MT =Examiner, full name recorded by research group Instrument Type: Seahorse XF96 analyzer (Agilent Technologies, CA, USA) Software for data generation: Wave 2.6.0 Information regarding all further metadata: Dataset number 2: Cells = MDA-MB-231 Repetitions: 1-5 1: related protocols 2: data analysis Graphpad Prism Version 5.04, serial number: GPW5-101165-NHI-7193 and EXcel 2016 t-test, significance levels *p<0,05, **p<0,01, ***p<0,001, ns: not significant 1) related protocols related protocols The MDA-MB-231 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37oC and 5% CO2. For cell cultivation, RPMI-1640 supplemented with 10% FBS was used. In culture medium, 10 mM HEPES buffer pH 7.4 and a mixture of 100 U/ml penicillin/100 ug/ml streptomycin (BioConcept Ltd.) was added. Passage number was between 8-22. Measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed in real-time with the Seahorse XF96 analyzer (Agilent Technologies, CA, USA). The different parameters of mitochondrial respiration were evaluated using the Seahorse XF Cell Mito Stress Test, according to the manufacturers instructions. Cells were transfected 48 h prior to the assays with siRNAs and seeded in 6 replicates per sample on a Seahorse XF96 V3 PS Cell Culture Microplate (Agilent Technologies). In this assay, baseline cellular OCR was measured, representing both mitochondrial and non-mitochondrial respiration. The proportion of mitochondrial OCR linked with ATP production is measured after addition of the ATPase inhibitor oligomycin at a concentration of 1ul. Subsequently, 0.5uM of the uncoupling agent FCCP is added, which collapses the electron gradient across the mitochondrial membrane and leads to a compensatory maximal oxygen consumption from the mitochondria, thereby enabling measurement of maximal respiration. The spare respiratory capacity is calculated by deduction of the basal mitochondrial respiration from this value. Finally, a mixture of 0.5 uM of antimycin and 0.5 uM rotenone is added, inhibiting function of complex III and I, and thus, completely shutting down mitochondrial oxygen consumption. This allows for calculation of non-mitochondrial OCR. Deduction of this value from that obtained after oligomycin treatment yields the portion of OCR resulting from proton leak. Basal respiration is the initial OCR value excluding non-mitochodrial respiration. Eto or Thio were added at a concentration of 40 ul 15 min prior to the commencement of the assay. Following the measurements, values were normalized to the protein content of each sample using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Data was analyzed employing the Wave 2.6.0 software. 2) data analysis ;Basal;;;;;;Maximal;;;;;;Spare;;;;;;ATP production;;;;; ;ctrl;;Eto;;Thio;;ctrl;;Eto;;Thio;;ctrl;;Eto;;Thio;;ctrl;;Eto;;Thio; ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 Exp.1;45.07;51.78;23.69;23.84;40.92;47.72;103.24;118.40;56.42;74.66;16.69;35.82;58.17;66.62;32.73;50.82;0.00;0.00;40.52;42.47;14.28;14.03;31.09;19.76 Exp.2;66.22;67.61;37.08;34.98;87.36;86.94;143.46;136.87;84.89;77.67;55.16;55.58;77.25;69.25;47.81;42.69;0.00;0.00;54.11;57.20;19.57;19.36;52.07;51.20 Exp.3;58.88;53.40;35.04;33.84;71.50;61.72;127.28;106.51;69.99;71.86;75.87;48.94;68.40;53.12;34.96;38.02;4.37;0.00;48.87;32.51;22.28;20.52;36.69;31.27 Exp.4;55.53;44.51;;;;;117.3256226;65.52695465;;;;;61.79502869;21.01995468;;;;;45.05;37.81;;;; Exp.5;53.87487793;50.90860748;24.51820564;28.53862953;59.58522415;59.89183807;90.74704742;60.70275116;57.33169556;51.80709076;75.34054565;66.68286133;36.87216949;9.794145584;32.81349564;23.26845932;15.7553215;6.79101944;47.46614075;45.2237587;15.04419136;19.36361313;31.78801727;31.85749054 ;;;;;;;;;;;;;;;;;;;;;;;; Avg;55.91;53.64;30.08;30.30;64.84;64.07;116.41;97.60;67.16;69.00;55.77;51.75;60.50;43.96;37.08;38.70;5.03;1.70;47.20;43.04;17.79;18.32;37.91;33.52 SD;6.883259599;7.610310297;6.026891611;4.449743563;16.96440558;14.25962643;18.34701278;29.8127984;11.55729591;10.13734631;24.05497144;11.1742256;13.4778416;24.20942742;6.260923722;10.01805793;6.443428494;2.940597676;4.47075048;8.291666384;3.287675629;2.521813325;8.454393431;11.28758517 t-test mock vs 17?-HSD12;0.669661219;;0.961031912;;0.95385711;;0.319981231;;0.842615373;;0.805527883;;0.275876735;;0.821578599;;0.458709582;;0.410525137;;0.834914209;;0.61075337; ;;;;;;;;;;;;;;;;;;;;;;;; t-test Eto or Thio vs ctrl;;;0.001240306;0.001670971;0.441410727;0.310545722;;;0.003614785;0.132285;0.01290882;0.034036851;;;0.022289316;0.709391;0.000346044;0.024332657;;;2.10575E-05;0.002540;0.154361699;0.269822264 ;;;;;;;;;;;;;;;;;;;;;;;; ;Proton leak;;;;;;Non-Mitochondrial ;;;;;;;;;;;;;;;;; ;ctrl;;Eto;;Thio;;ctrl;;Eto;;Thio;;;;;;;;;;;;; ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;mock ;17B12 ;;;;;;;;;;;; Exp.1;4.552451134;9.31391716;9.408061981;9.816984177;9.826303482;27.96307945;14.78343296;24.72872353;14.30473518;11.97548199;27.40607262;15.60146427;;;;;;;;;;;; Exp.2;12.1054163;10.41196918;17.5044136;15.62265015;35.2880249;35.74446869;25.28303909;30.66362;15.97154236;18.78308105;18.50377274;17.50814056;;;;;;;;;;;; Exp.3;10.01272774;20.88678741;12.75106049;13.31851959;34.80820465;30.44203568;23.19748688;19.24501228;18.44892311;16.16407394;18.1246376;14.37285709;;;;;;;;;;;; Exp.4;10.48261261;6.693041325;;;;;18.73040009;20.91952515;;;;;;;;;;;;;;;; Exp.5;6.408733368;5.684845448;9.474014282;9.175016403;27.79720688;28.03434753;23.84392548;21.78172302;22.53095627;18.0737648;14.86243534;16.17718506;;;;;;;;;;;; ;;;;;;;;;;;;;;;;;;;;;;;; Avg;8.71;10.60;12.28;11.98;26.93;30.55;21.17;23.47;17.81;16.25;19.72;15.91;;;;;;;;;;;; SD;2.791025583;5.420893146;3.302949527;2.627121136;10.31032457;3.162861124;3.870853392;4.01343369;3.096869125;2.646766989;4.655533403;1.127313979;;;;;;;;;;;; t-test mock vs 17?-HSD12;0.559013624;;0.905888427;;0.596165509;;0.433313614;;0.531128469;;0.253204514;;;;;;;;;;;;; ;;;;;;;;;;;;;;;;;;;;;;;; t-test Eto or Thio vs ctrl;;;0.18287714;0.671011448;0.051359886;0.000602667;;;0.243903953;0.024675;0.678850989;0.016924607;;;;;;;;;;;;