S adenosylmethionine sam

Human prostate cancer cell lines PC-3 and DU145 were purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig. The cells were seeded in 25 cm² culture flasks in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 1.2% penicillin/streptomycin as described before [19 ] (PAN-Biotech GmbH, Aidenbach, Germany). Subsequently, they were treated either with vehicle (0.005 M H₂SO₄) or 200 µm of S-adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA). The most effective concentration of SAM for both cell lines was determined in a previous study [19 ]. Treatment with vehicle or SAM was carried out by direct addition to the regular growth medium under sterile conditions. The medium was changed every other day.

Bisulfite conversion of genomic DNA from TICs was carried out using the Epitect Bisulfite kit (QIAGEN, USA). Briefly, 500 ng DNA from TICs was added to 200 μl sterile PCR tube. The samples were mixed up with 85 μl bisulphite conversion reagent (bisulphite mix), 85 μl DNA protect buffer, and RNase-free water making up total volume of 140 μl. The samples were then processed according to manufacturer’s instructions. For positive controls, fully methylated, positive controls were generated by incubating genomic DNA with DNA methyltransferases in the presence of S-Adenosyl methionine (SAM) (New England bio lab, USA) for 2 h at 37 °C prior to bisulfite conversion.

Overnight broth cultures of H. pylori PMSS1 were subcultured and grown until mid-log phase. The bacterial cell pellet was resuspended in 5× volume of extraction buffer containing 20 mM Tris-acetate (pH 7.9), 50 mM potassium acetate, 5 mM Na₂EDTA, 1 mM dithiothreitol (DTT), and a protease inhibitor cocktail (cOmplete, Mini, EDTA-free from Sigma-Aldrich, St. Louis, MO). The bacterial cell suspension was placed in a screw cap microcentrifuge tube containing 0.1-mm glass beads (Biospec Products, Bartlesville, OK). The tube was oscillated on a bead beater (Mini-Beadbeater-24 from Biospec Products) and then centrifuged at 15,000g for 5 minutes at 4 °C. The supernatant was removed, and protein concentration of the supernatant was determined using the Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, Hercules, CA). Linear DNA construct was combined with the supernatant (300–400 μg of protein) in a reaction containing extraction buffer (without protease inhibitor) and 200 μM S-adenosylmethionine (SAM) (New England BioLabs, Ipswich, MA), and incubated for 1 hour at 37 °C. Linear DNA construct was purified using the Zymo DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA), and transformed into PMSS1.

DBP and MBP were purchased from Tokyo Kasei Kogyo Co Ltd. (Tokyo, Japan). Human chorionic gonadotrophin (hCG) and forskolin were obtained from Sigma (St. Louis, MO, USA). RPMI 1640 medium, fetal bovine serum (FBS), streptomycin sulfate, antibiotic penicillin G sodium (10,000 U/ml), and phosphate-buffered saline with Ca²⁺ and Mg²⁺ were obtained from Gibco (Grand Island, NY, USA). S-adenosylmethionine (SAM) was purchased from New England BioLabs (Ipswich, MA, USA). All other chemicals used were of analytical grade.

Histone methyltransferase (HMTase) assay was performed using immunoprecipitated EZH2 and its co-precipitating proteins. Briefly, native chromatin from each sample was extracted as described for ChIP-qPCR below, without fixation. Appropriate amounts of solubilized chromatin were incubated with 2 μg of anti-EZH2 antibody (Active Motif, Carlsbad, CA, USA) and 20 μl of Dynabeads M-280 Sheep Anti-Mouse IgG (Life Technologies, Carlsbad, CA, USA) at 4°C for 4 h. The beads were washed two times with ChIP buffer (10 mM Tris-HCl [pH 8.0], 200 mM KCl, 1 mM CaCl₂, and 0.5% NP-40), two times with Wash buffer (10 mM Tris-HCl [pH 8.0], 500 mM KCl, 1 mM CaCl₂, and 0.5% NP-40), and once with HMTase buffer (20 mM phosphate buffer [pH 7.4] and 0.05% Tween-20). The immunoprecipitated protein was incubated at 30°C for 3 h in 30 μl of HMTase buffer containing 1 μg of recombinant histone H3.1 protein (New England Biolabs, Ipswich, MA, USA) as substrate and 40 μM S-adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA, USA) as the methyl donor. The reaction was stopped by addition of 10 μL of 4× Laemmli sample buffer. Proteins were separated by SDS-PAGE and subjected to western blotting. Each western blotting signal was quantified using the ImageJ software as described above, and the H3K27me3 level was normalized to the immunoprecipitated EZH2 protein signal.

Both plasmids, pCpGL-PLIN and pCpGL-basic (no insert), were methylated using SssI methyltransferase (New England Biolabs, Hitchin, UK) according to the manufacturer’s recommendation. In brief, 10–15 µg of plasmid DNA was incubated with or without SssI methyltransferase (20 U/µl; 2 U/µg DNA) in the presence of 640 µM S-Adenosylmethionine (SAM) (New England Biolabs) for four hours at 37 °C, with another 640 µM SAM being added after the first two hours of incubation. Plasmid DNA was purified using QIAquick PCR purification kit (Qiagen). Methylation of plasmid DNA was controlled by digestion using methylation sensitive restriction enzyme HpaII (Thermo Fisher Scientific) for four hours at 37 °C.

N6-methyladenine (6mA)-modified (at the RGATCY motif) MB probe was chemically synthesized using 5′-dimethoxytrityl-N6-methyl-2′-deoxyadenosine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research, Sterling, VA, USA) by FASMAC.
Alternatively, the MB probe in a final concentration of 50 µM was mixed with 1.12 mM S-adenosylmethionine (SAM) (New England Biolabs), 1× Dam methyltransferase reaction buffer (New England Biolabs), 16 U E. coli Dam methyltransferase (New England Biolabs) or 2 µL of the NRS3_07 CFPS solution, and nuclease-free H₂O to a total volume of 20 µL. The methylation reaction was carried out at 37°C for 6 h.