Ethylene glycol bis

Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (Sigma-Aldrich, St-Louis, MO, USA), Dithiothreitol (DTT) (Sigma-Aldrich), Lithium chloride (LiCl) (Sigma-Aldrich), N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) (Sigma-Aldrich), (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1 (link),6 (link)]benzodiazocine-10-carboxylic acid methyl ester (K252a) (Sigma-Aldrich), (2R)-2-1-butanol (Roscovitine) (Sigma-Aldrich), 4,5,6,7-Tetrabromo-2-azabenzimidazole (TBB) (Sigma-Aldrich), 1-(7-methoxyquinolin-4-yl)-3-(6-(trifluoromethyl)pyridin-2-yl)urea (A-1070722) (Sigma-Aldrich), cyclosporine A (Sigma-Aldrich), N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (Saracatinib) (Selleck Chemicals, Houston TX), (5S,6R,7R,9R)-6-methoxy-5-methyl-7-(methylamino)-6,7,8,9,15,16-hexahydro-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-14(5h)-one (STS) (ab120056; Abcam, Cambridge, MA, USA), Z-Asp-2,6-Dichlorobenzoyloxymethyl Ketone (Z-DCB) (Cayman Chemical, Ann Arbor Michigan) and okadaic acid (Cell Signaling Technology, Danvers, MA). SNJ-1945 was a gift from (Senju Pharmaceutical Co. Ltd., Kobe, Japan) (Table 1).

Test compounds were purchased from commercial sources. Caffeic acid and rosmarinic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Salvianolic acids A and B (Sal A and Sal B) were purchased from TAUTO (Shanghai Tauto Biotech Co. Ltd., Shanghai, China) and Sal C was purchased from Herbest (Baoji Herbest Bio-Tech Co. Ltd., Baoji, China). All the tested compounds were of HPLC grade with ≥98% purity. Human recombinant GSK-3β was obtained from Prospec (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel). GSM substrate mimicking glycogen muscle synthase was obtained from Merck Millipore (Millipore Corporation, Temecula, CA, USA) and a Kinase-Glo kit was obtained from Promega (Promega Corporation, Madison, WI, USA). Adenosine 5-triphosphate (ATP) disodium salt hydrate, ammonium hydroxide, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), ammonium acetate, ethylene glycol-bis(-aminoethylether)-N,N,N′,N′-tetraacetic acid tetrasodium salt (EGTA), formic acid, ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), 3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione, and magnesium acetate tetrahydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were purchased from E. Merck, Fluka (Rupert-Mayer-Str., Munich, Germany) and Sigma-Aldrich unless otherwise stated.

ChIP was performed as described29 (link) with minor modifications. For H3K9me3 ChIP, cells were crosslinked in phosphate-buffered saline (PBS) containing 1% formaldehyde (28908, ThermoFisher Scientific) for 5 min. For p65 ChIP, cells were first crosslinked in PBS containing 2 mM ethylene glycol-bis (E3257, Sigma-Aldrich) for 30 min, and then in PBS containing 1% formaldehyde for another 10 min. Chromatin was sheared by Sonifier SLPe40 (Branson Ultrasonics, Danbury, CT) and the average DNA size (approximate 400 bp) was confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Sonicated nuclear extracts were immunoprecipitated with primary antibodies listed in Supplementary Table S1. The purified DNA was quantified with quantitative real-time PCR using the primer sequences listed in Supplementary Table S4. The results are presented as a percent input value.

Antibodies [anti-c-Myc (Santa Cruz Biotechnology, Dallas, Texas, sc-40) and anti-HA (Santa Cruz Biotechnology, sc-7392)]
Complete protease cocktail inhibitor (Roche, cat. no. 04693159001)
Dithiothreitol (DTT) (Sigma, cat. no. D-9779)
Ethylenediaminetetraacetic acid (EDTA) (Sigma, cat. no. E-4884)
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma, cat. no. E-3889)
Ethanol (Sigma, cat. no. E-7023)
Formaldehyde 37% (Sigma, cat. no. F-8775)
Glycine (Sigma, cat. no. 50046)
Glycogen (Roche, cat. no. 10901393001)
HEPES (Sigma, cat. no. H-3375)
Lithium chloride (Sigma, cat. no. L-4408)
Nonidet P-40 (NP-40) (see Comment, below)
Proteinase K (Ambion, cat. no. AM2546)
Pre-equilibrated salmon sperm DNA/protein A agarose beads (Millipore, cat. no. 16-157)
Sodium acetate (Sigma, cat. no. 127-09-3)
Sodium chloride (Sigma, cat. no. 7647-14-5)
Sodium deoxycholate (Sigma, cat. no. D-6750)
Sodium dodecyl sulfate (Sigma, cat. no. L-6026)
Tris (Sigma, cat. no. 93362)
Triton X-100 (Sigma, cat. no. T-8787)

Total protein concentration was determined based on the Bradford method. To extract proteins, the same tissue powder was used as for the TAG content measurement. One hundred milligram of this powder was homogenized in a lysis buffer [50 mM HEPES (Carl Roth GmbH, Karlsruhe, Germany), 4 mM ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (Sigma-Aldrich), 10 mM EDTA (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich), 100 mM β-glycerol phosphate (Sigma-Aldrich), 15 mM sodium pyrophosphate (Sigma-Aldrich), 5 mM sodium orthovanadate (Sigma-Aldrich), 2.5 mM sodium fluoride (Sigma-Aldrich) and a protease inhibitor cocktail (CompleteMini, Roche Diagnostics GmbH, Mannheim, Germany)] with a FastPrep-24 tissue homogenizer (MPI Biomedicals). Protein extracts were centrifuged at 10,000 g for 5 min at 4°C. Protein concentration was measured in the corresponding fraction by using Bradford reagent (Serva Electrophoresis GmbH) according to the manufacturer’s protocol. Finally, measured protein concentrations were corrected for wet weight of the sample and results were expressed as mg total protein per g tissue.

2,4-Dinitrophenol (DNP), chloroform, dimethyl sulfoxide (DMSO), 1,2-dioleoyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cardiolipin (CL), sodium sulfate (Na₂SO₄), 2-(N-morpholino) ethanesulfonic acid (MES), tris(hydroxymethyl)-aminomethane (Tris), adenosine 5′-triphosphate (ATP), guanosine 5′-triphosphate (GTP), carboxyatractyloside (CATR), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), N-lauroylsarcosine, Triton X-114, dithiothreitol (DTT), bovine serum albumin (BSA), sucrose, 3-(N-morpholino) propanesulfonic acid (MOPS), rotenone, and safranin O were purchased from Sigma-Aldrich (Vienna, Austria). Arachidonic acid (AA) was purchased from Larodan (Solna, Sweden), n-octylpolyoxyethylene was purchased from BACHEM (Bubendorf, Switzerland), and hydroxyapatite was purchased from Bio-Rad Laboratories (Hercules, CA, USA).