File Name: 10.1194/jlr.M092908_kinetic values (Km and vmaxapp) described in section 3.1

Technique/Assay: Number 4 = LC-MS/MS
SK and KB =Examiner, full name recorded by research group
Instrument Type: Agilent 1290 Infinity UHPLC binary solvent delivery system equipped with a column oven and a temperature controlled auto sampler, coupled to an Agilent 6490 triple quadrupole mass spectrometer with a jet stream electrospray ionization interface (AJS-ESI) (Agilent Technologies, California, USA) 
Software version for data generation:Mass Hunter Workstation Acquisition Software Version 07.01 SP1 and MassHunter Workstation Software Quantitative Analysis Version B.07.00 /Build 7.0457.0 (Agilent Technologies).

Also see: 31003A-179400_20181120_KB_27Oxysterol_4_6_M_2: LC-MS_Acquisition report.Pdf

Information regarding all further meta-data: 


1)  related protocols cell culture, enzyme kinetic determination
2)  Analyte extraction Protocol LC-MS/MS
3) HEK-293 cells stably expressing 11b-HSD1-H6PDH_construction_publication.pdf
4) microsome preparation_publication.pdf
5) Data analysis (Graphpad Prism Version 5.04)
6) protein determination 
7) chemicals and reagents
8) protein abbreviation

1) related protocols cell culture, enzyme kinetic determination:
Medium HEK-293 cells
Dulbeccos modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 4.5 g/L glucose, 100 U/mL penicillin/streptomycin, 2mM L-glutamine, 10mM HEPES, pH 7.4, and 1% MEM non-essential amino acid solution
Preparation of microsomes
The preparation of microsomes was conducted as reported earlier (31003A-179400_20181120-21-22_KB_27Oxysterol_4T_1_M_5) with minor modifications. Briefly, HEK-293 cells stably expressing
human 11b-HSD1 and H6PDH (31003A-179400_20181120-21-22_KB_27Oxysterol_4T_1_M_4) were grown until they reached confluence. The cells were washed twice with ice-cold PBS, resuspended to a final 
concentration of ~2x10^7 cells/mL in ice-cold buffer A (50mM Tris pH 7.5, 1mM EDTA, 100mM NaCl) and homogenated with 20 strokes in glass potter on ice. After sonication, the homogenate was centrifuged at 9000 x g, 4 oC for 30 min. 
The supernatant was transferred for ultracentrifugation at 105'000 x g, 4 oC for 70 min. Then, the pellet was resuspended in buffer B (20mM Tris pH 7.5, 1mM EDTA, 10% glycerol) and the ultracentrifugation step repeated. The pellet
containing the microsomal fraction was resuspended in buffer B and the total protein concentration determined by using BCA assay. The microsomes were immediately frozen on dry ice and stored at -80 oC.
Determination of apparent KM and vmax values
Enzyme kinetics were assessed in a total assay volume of 50 uL. The incubation time and the amount of microsomes was adapted to the linear range of product formation. For 11b-HSD1, 1-20 ug of microsomes were incubated for 20 min in TS2 buffer (100mM NaCl, 1mM
EGTA, 1mM EDTA, 1mM MgCl2, 250mM sucrose, 20mM Tris-HCl, pH 7.4), 1mM NADPH, 2mM glucose-6-phosphate (G6P), 0.1 Unit glucose-6-phosphate dehydrogenase (G6PD) (from S. cerevisiae) and various concentrations (62.5 - 2000 nM) of 7k27OHC. 


2) Analyte extraction Protocol LC-MS/MS:
Quantification of oxysterols was performed by UHPLC-MS/MS. For this purpose, microsomes were mixed with ice-cold ethylacetate containing 100 nM deuterium-labeled 7b27OHC as internal standard 
and 1.5% (v/v) of a standard solution of BHT and TPP as antioxidants. Then, the samples were incubated in a rotating mixer for 15 min at 4 oC, centrifuged for 2 min, 16'000 x g at 4 oC and the upper ethylacetate phase transferred
into a new tube. The liquid-liquid extraction step was repeated, the ethylacetate phase combined with the one from the former step and evaporated to dryness under nitrogen. The samples were reconstituted in 50 uL 
methanol:ultra-pure water (1:1, v/v), centrifuged at 4 oC, 16'000 x g for 10 min and the supernatant analyzed.


3) Inhibition of 11b-hydroxysteroid dehydrogenase type 1 by plant extracts used as traditional antidiabetic medicines
Christel Gumy et al.
Fitoterapia 80 (2009) 200-205
doi:10.1016/j.fitote.2009.01.009

4) 11beta-Hydroxysteroid dehydrogenase 1 reductase activity is dependent on a highratio of NADPH/NADP+ and is stimulated by extracellular glucose
Anna A. Dzyakanchuk et al.
Molecular and Cellular Endocrinology 301 (2009) 137-141
doi:10.1016/j.mce.2008.08.009

5) kinetic analysis 11bHSD1 and 7k27OHC described in section 3.1	
	
data used	
31003A-179400_20181120_KB_27Oxysterol_4_6_1	
31003A-179400_20181121_KB_27Oxysterol_4_6_2	
31003A-179400_20181122_KB_27Oxysterol_4_6_3	
	
concentration (nM);velocity velocity calcualted as v = [nmoles/(h*mg protein)]	
62.5;2.158674;2.189554;2.721907;;with time = 0.33 h	
125;3.148513;3.75;3.908215;;"protein amount: 62.5 and 125 nM substrate: 0.001 mg; 250 nM: 0.0025 mg; 500 nM: 0.005 mg; 1000 nM: 0.01 mg and 2000 nM 0.02 mg"	
250;2.875176;4.151283;3.68488;;total assay volume 50 uL	
500;4.359532;4.856349;3.570844;;	
1000;4.550442;3.898958;3.905998;;	
2000;4.432118;3.135577;4.14953;;	

Michaelis Menten analysis with GraphPad Prism Version 5.04 serial number: GPW5-101165-NHI-7193
	
Michaelis-Menten	
Best-fit values	
Vmax;4.272	
Km;39.12	
Std. Error	
Vmax;0.2099	
Km;12.29
95% Confidence Intervals
Vmax;3.827 to 4.717	
Km;13.06 to 65.18	
Goodness of Fit
Degrees of Freedom;16	
R square;0.5637
Absolute Sum of Squares;4.549	
Sy.x;0.5332	
Constraints	
Km;Km > 0.0	
Number of points	
Analyzed;18	

7) Chemicals and reagents

balance	
Mettler Toledo	
XPE105	
Delta Range Max 41g/120g d=0.01mg/0.1mg

Name;abbrevation;CAS;supplier;stock prepared									
7-keto-27-hydroxycholesterol or 3b27-dihydroxy-5-cholesten-7-one;7k27OHC;240129-30-0;Avanti Polar Lipids Inc. (Alabaster AL);1 mM in DMSO						
7b27-dihydroxycholesterol;7b27OHC;240129-43-5;Avanti Polar Lipids	 Inc. (Alabaster AL);1 mM in DMSO						
7a27-dihydroxycholesterol;7a27OHC;4725-24-0;Avanti Polar Lipids	 Inc. (Alabaster AL);1 mM in DMSO						
7b27-dihydroxycholesterol-d6;7b27OHC-d6;2260669-26-7;Avanti Polar Lipids	 Inc. (Alabaster AL);0.5 mg/mL in MeOH						
1,1,1,3,3,3-Hexafluoro-2-(2-fluoro-4-((4-(pyridin-4-ylmethyl)piperazin-1-yl)methyl)-[1,1-biphenyl]-4-yl)propan-2-ol, ;2-Fluoro-4?-[[4-(4-pyridinylmethyl)-1-piperazinyl]methyl]-a,a-bis(trifluoromethyl)-[1,1?-biphenyl]-4-methanol, ML310;SR2211;1359164-11-6;Cayman Chemicals (Ann Arbor	 MI);10 mM in DMSO								
5H-1,2,4-triazolo(4	3-a)azepine 6,7,8,9-tetrahydro-3-tricyclo(3-3-1-13-7)dec-1-yl also known as Merck-544;T0504;;Enamine (Kiev Ukraine).;10 mM in DMSO
[1,2-3H]-cortisone;-;-;American Radiolabeled Chemicals (St.Louis MO);50-60 Ci/mmol 1.85-2.22 TBq/mmol in EtOH -->1 mCi/ml							
hydrocortisone  [1	2-3H];[1	2,6,7-3H]-cortisol ;-;American Radiolabeled Chemicals (St.Louis MO);50-60 Ci/mmol 1.85-2.22 TBq/mmol in EtOH --> 1 mCi/mL				
[1,2,6,7-3H] corticosterone;-;-;American Radiolabeled Chemicals (St.Louis MO);70-100 Ci/mmol 2.59-3.7 TBq/mmol in EtOH --> 1 mCi/mL					
7b-hydroxycholesterol or cholest-5-en-3b7b-diol;7bOHC;566-27-8;Sigma Aldrich (Buchs	 Switzerland);1 mM in DMSO							
7-ketocholesterol or 3b hydroxy-5-cholestene-7-one;7kC;566-28-9;Sigma Aldrich (Buchs	 Switzerland);1 mM in DMSO								
cortisone;-;53-06-5;Sigma Aldrich (Buchs Switzerland);10 mM in DMSO								
cortisol;-;50-23-7;Sigma Aldrich (Buchs	 Switzerland);10 mM in DMSO								
18b-Glycyrrhetinic acid;GA;471-53-4;Sigma Aldrich (Buchs	 Switzerland);10 mM in DMSO								
butylated hydroxytoluene;BHT;128-37-0;Sigma Aldrich (Buchs Switzerland);10 mg BHT								
triphenylphosphine;TPP;791-28-6;Sigma Aldrich (Buchs Switzerland);and 25 mg TPP in 10 mL ethanol								
b-Nicotinamide adenine dinucleotide 2?-phosphate reduced tetrasodium salt;;2646-71-1 (anhydrous);Sigma Aldrich (Buchs Switzerland);25 mM in nuclease free deonized water								
b-Nicotinamide adenine dinucleotide;;53-84-9;Sigma Aldrich (Buchs Switzerland);25 mM in nuclease free deonized water								
glucose-6-phosphate;;54010-71-8;Sigma Aldrich (Buchs Switzerland);50 mM in nuclease free deonized water								
glucose-6-phosphate dehydrogenase (from S. cerevisiae);;9001-40-5;Sigma Aldrich (Buchs Switzerland);1mg/mL nuclease free deonized water								
NaCl;;7647-14-5;Sigma Aldrich (Buchs	 Switzerland);depending on the buffer								
EGTA;;67-42-5;Sigma Aldrich (Buchs	 Switzerland);depending on the buffer								
EDTA;;K36169917;Sigma Aldrich (Buchs Switzerland);depending on the buffer								
MgCl2 Hexahydrate;;7791-18-6;Sigma Aldrich (Buchs Switzerland);depending on the buffer								
Sucrose;;57-30-1;Sigma Aldrich (Buchs Switzerland);depending on the buffer								
Tris HCl;;1185-53-1;Sigma Aldrich (Buchs Switzerland);depending on the buffer								
doxycycline hyclate;;24390-14-5;Sigma Aldrich (Buchs Switzerland);10 mM in DMSO


8) Proteins:

Protein;Gene;species;Abbrevation used
hydroxysteroid 11-beta dehydrogenase 1;HSD11B1;human;11b-HSD1
hydroxysteroid 11-beta dehydrogenase 1;Hsd11b1;mouse;11b-hsd1
hydroxysteroid 11-beta dehydrogenase 2;HSD11B2;human;11b-HSD2
hydroxysteroid 11-beta dehydrogenase 2;Hsd11b2;mouse;11b-hsd2
hexose-6-phosphate dehydrogenase/glucose 1-dehydrogenase;H6PD;human;H6PD
cytochrome p 450 27A1;CYP27A1;human;CYP27A1
